Method of treating urea cycle disorder by interfering with glucagon receptor signaling

Abstract

To provide methods of treating a subject with hyperammonemia or a urea cycle disorder.SOLUTION: The methods comprise administering a therapeutic amount of a glucagon signaling pathway inhibitor to a subject in need thereof so that ammonia levels are lowered or amino acid metabolism enzymes are downregulated, or the effects of conditions or diseases characterized by hyperammonemia are lowered, or at least one symptom or complication associated with the condition or disease is alleviated or reduced in severity. The glucagon signaling pathway inhibitor can be a small molecule inhibitor of the signaling pathway, an antisense inhibitor of the signaling pathway, shRNA, siRNA, a GCG neutralizing monoclonal antibody, a GCGR antagonist, a peptide inhibitor of the signaling pathway, a DARPin, a Spiegelmer, an aptamer, engineered Fn type-III domains, and the like.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to methods of using glucagon (GCG) inhibitors or glucagon receptor (GCGR) antagonists to treat urea cycle disorders and / or hyperammonemia, and / or methods of reducing the therapeutic dose of sodium phenylbutyrate or sodium benzoate in a subject in need thereof.

[0002] Sequence Listing An official copy of the Sequence Listing was submitted electronically via EFS-Web as an ASCII formatted Sequence Listing contemporaneously herewith. The filename of the copy is "10366WO01_US_SEQ_LIST_ST25," has a creation date of August 21, 2018, and is approximately 116 KB in size. The Sequence Listing contained in this ASCII format document is a part of the present specification and is incorporated herein by reference in its entirety. [Background technology]

[0003] background Glucagon is a 29-residue polypeptide hormone that, in conjunction with insulin, mediates homeostatic regulation of blood glucose levels. Glucagon acts primarily by stimulating certain cells, such as hepatocytes, to release glucose to maintain normal blood glucose levels when blood glucose levels fall. Glucagon's action is opposite to that of insulin, which stimulates cells to take up and store glucose whenever blood glucose levels rise. Glucagon is produced by alpha cells in the pancreas, while insulin is secreted by adjacent beta cells. The glucagon receptor is a member of the class B G protein-coupled family of receptors and is activated by glucagon binding. Glucagon receptors are primarily expressed in the liver and kidney.

[0004] The action of glucagon can be suppressed by providing an antagonist such as a small molecule inhibitor, shRNA, siRNA, GCG antibody, or GCGR antibody, as described herein.Anti-GCG antibody is described in, for example, U.S. Patent Nos. 4,206,199, 4,221,777, 4,423,034, 4,272,433, 4,407,965, 5,712,105, and PCT Publications WO2007 / 124463 and WO2013 / 081993. Anti-GCGR antibodies are described in U.S. Patent Nos. 5,770,445, 7,947,809, and 8,545,847; European Patent Application No. EP2074149A2; European Patent No. EP0658200B1; U.S. Patent Publications 2009 / 0041784; 2009 / 0252727; and 2011 / 0223160; and PCT Publication WO2008 / 036341. Small molecule inhibitors of GCG or GCGR are described, for example, in WO07 / 47676; WO06 / 86488; WO05 / 123688; WO05 / 121097; WO06 / 14618; WO08 / 42223; WO08 / 98244; WO2010 / 98948; US20110306624; WO2010 / 98994; WO2010 / 88061; WO2010 / 71750; WO2010 / 30722; WO06 / 104826; WO05 / 65680; WO06 / 102067; WO06 / 17055; WO2011 / 07722; or WO09 / 140342. Glucagon inhibition affects the GCG / GCGR downstream signaling pathway, inhibiting cAMP and PKA.

[0005] Urea cycle disorders (UCDs) result from genetic mutations that cause defects in the metabolism of nitrogen, which is produced by the breakdown of proteins and other nitrogen-containing compounds. Deficiencies of N-acetylglutamate synthetase (NAGS), carbamoylphosphate synthetase I (CPSI), ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASS, also known as citrullinemia I), citrine (citrullinemia II), argininosuccinate lyase (argininosuccinic aciduria), arginase (hyperargininemia), and ornithine translocase (HHH syndrome) are associated with these disorders. However, the onset of acute hyperammonemia varies, as some disorders affect newborns and others adults; some disorders, such as citrullinemia II, can present in either newborns or adults. Amino acid metabolic enzymes are upstream of the urea cycle and contribute to ammonia production through the breakdown of amino acids. The enzymes involved include cystathionase (CTH), serine dehydratase (SDS), ornithine aminotransferase (OAT), and glutaminase 2 (GLS-2).

[0006] Urea cycle disorders and hyperammonemia cause a variety of symptoms, but typically, without treatment, they result in intellectual and developmental disabilities and ultimately death. Available treatment options include dietary protein restriction and nitrogen wasting using phenylbutyrate, but ultimately many subjects with hyperammonemia and / or urea cycle disorders require liver transplantation.

[0007] In the absence of effective therapies for treating or slowing the progression of severe hyperammonemia and / or urea cycle disorders, i.e., for prolonging the survival and / or improving the quality of life of subjects with hyperammonemia and / or urea cycle disorders, there is a need to identify and explore the use of other agents for treating these diseases, such as glucagon signaling pathway inhibitors and antagonists, as described herein. Summary of the Invention

[0008] overview Provided herein is a method for treating a subject with a condition or disease characterized by hyperammonemia by administering a glucagon signaling pathway antagonist, such as a GCG inhibitor or a GCGR antagonist, or a pharmaceutical composition comprising a GCG inhibitor or a GCGR antagonist. A GCG inhibitor or a GCGR antagonist is a compound that can block or inhibit the glucagon receptor signaling pathway. Antagonists can take the form of small molecule inhibitors, shRNA, siRNA, peptide inhibitors, CRISPR technology (Clustered regularly interspaced short palindromic repeats; CRISPR technology can generate GCGR knockdown or deletion of regulatory sequences that affect GCGR activity), antisense inhibitors, DARPin, and GCG or GCGR neutralizing monoclonal antibodies. The glucagon signaling pathway antagonist can be administered alone, in a pharmaceutical composition, or in conjunction with one or more therapeutic agents, supplements, or therapeutic procedures useful for treating a condition or disease associated with hyperammonemia, or useful for treating one or more symptoms associated with a urea cycle disorder, or useful in lowering blood ammonia levels in a subject having a condition or disease associated with a urea cycle disorder.

[0009] In some embodiments, the method comprises administering a therapeutically effective amount of a composition containing a glucagon signaling pathway antagonist to a subject with hyperammonemia to reduce serum ammonia levels, or to reduce the effects of the condition or disease, or to alleviate or reduce the severity of at least one symptom or complication associated with the condition or disease. In some aspects, the hyperammonemia is congenital hyperammonemia. For example, congenital hyperammonemia can be caused by a deficiency of a urea cycle enzyme selected from the group consisting of carbamyl phosphate synthetase (CPS1), N-acetylglutamate synthetase (NAGS), ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL), and arginase (AR1), or a deficiency of a urea cycle transporter, such as ornithine translocase (ORNT1) and citrin. Congenital hyperammonemia can be caused by methylmalonic aciduria, propionic aciduria, or isovaleric aciduria. Congenital hyperammonemia can be caused by medium-chain acyl-CoA dehydrogenase deficiency, multiple acyl-CoA dehydrogenase deficiency, carnitine palmitoyltransferase II deficiency, carnitine-acylcarnitine translocase, lysinuric protein intolerance, pyrroline-5-carboxylic acid synthetase deficiency, pyruvate carboxylase deficiency, ornithine aminotransferase deficiency, carbonic anhydrase Va deficiency, hyperinsulinemia-hyperammonemia syndrome, mitochondrial disorders, and glutamine synthetase deficiency. In some embodiments, hyperammonemia is acquired. For example, hyperammonemia can be caused by liver disease and its complications, treatment with therapeutic agents (L-asparaginase or pegaspargase), 5-pentanoic acid, valproic acid, corticosteroids, or cyclophosphamide.

[0010] In some embodiments, the hyperammonemia is caused by herpes simplex infection, hepatitis B infection, or infection with a urease-producing organism.

[0011] In some aspects, hyperammonemia is caused by total parenteral nutrition (with relative arginine deficiency), L-asparaginase treatment, nutritional carnitine deficiency, cystoscopy with glycine-containing solutions, after lung / bone marrow transplantation, congenital vascular anomalies, or transient hyperammonemia in newborns.

[0012] Provided herein are methods for treating a subject having a urea cycle disorder, wherein the subject exhibits elevated levels of ammonia. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a composition comprising a glucagon signaling pathway antagonist.

[0013] Provided herein are methods for treating a subject having a urea cycle disorder, wherein the subject does not exhibit elevated levels of ammonia. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a composition comprising a glucagon signaling pathway antagonist.

[0014] Provided herein are methods for reducing blood ammonia levels, or for treating a condition or disease associated with or characterized in part by hyperammonemia, or for treating at least one symptom or complication associated with the condition or disease. In some embodiments, the methods comprise administering to a subject a therapeutically effective amount of a composition containing a glucagon signaling pathway antagonist such that blood ammonia levels are reduced, or the effects of the condition or disease are reduced, or at least one symptom or complication associated with the condition or disease is alleviated or reduced in severity.

[0015] In some embodiments, the glucagon signaling pathway antagonist is selected from small molecule inhibitors, shRNA, siRNA, peptide inhibitors, CRISPR technology (Clustered regularly interspaced short palindromic repeats, CRISPR technology can generate GCGR knockdown or deletion of regulatory sequences that affect GCGR activity), antisense inhibitors, DARPins, and GCG inhibitors or GCGR antagonists (e.g., neutralizing monoclonal antibodies).

[0016] In some embodiments, the GCGR antagonist can be an anti-GCGR antibody. The anti-GCGR antibody can inhibit or antagonize GCGR. The anti-GCGR antibody can inhibit or block the GCGR signaling pathway. In some embodiments, the GCG inhibitor can be an anti-GCG antibody. The anti-GCG antibody can inhibit the binding of GCG to GCGR.

[0017] In certain embodiments, the antibody or antigen-binding fragment specifically binds to hGCGR and comprises heavy and light chain CDR domains contained within a heavy and light chain sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 68, 70 / 78, 86 / 88, 90 / 98, 106 / 108, 110 / 118, 126 / 128, 130 / 138 and 146 / 148.

[0018] In certain embodiments, the antibody or antigen-binding fragment comprises the heavy and light chain CDR domains contained within the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 86 / 88.

[0019] In certain embodiments, the antibody or antigen-binding fragment comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 86 / 88.

[0020] In one embodiment, the human antibody or antigen-binding fragment of a human antibody that binds to hGCGR comprises a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 70, 86, 90, 106, 110, 126, 130, and 146, or a sequence substantially similar thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0021] In one embodiment, the human antibody or antigen-binding fragment of a human antibody that binds to hGCGR comprises a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 68, 78, 88, 98, 108, 118, 128, 138, and 148, or a sequence substantially similar thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0022] In certain embodiments, the human antibody or fragment thereof that binds to hGCGR comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 68, 70 / 78, 86 / 88, 90 / 98, 106 / 108, 110 / 118, 126 / 128, 130 / 138, and 146 / 148. In certain embodiments, the HCVR / LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 34 / 42, 70 / 78, 86 / 88, 110 / 118, and 126 / 128.

[0023] In certain embodiments, an isolated human antibody or antigen-binding fragment thereof that specifically binds to hGCGR comprises an HCVR comprising three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within an HCVR sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 70, 86, 90, 106, 110, 126, 130, and 146, and / or an LCVR comprising three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within an LCVR sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 68, 78, 88, 98, 108, 118, 128, 138, and 148.

[0024] In certain embodiments, the methods provided herein contemplate the use of an isolated human antibody or antigen-binding fragment thereof that binds hGCGR, comprising an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 24, 40, 56, 76, 96, 116, and 136, or a sequence substantially similar thereto with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and / or an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 32, 48, 64, 84, 104, 124, and 144, or a sequence substantially similar thereto with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0025] In one embodiment, the method provided herein comprises the step of: providing an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 20, 36, 52, 72, 92, 112 and 132, or a sequence substantially similar thereto with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 22, 38, 54, 74, 94, 114 and 134, or a sequence substantially similar thereto with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; The use of antibodies or fragments thereof is contemplated, which further comprise an LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 28, 44, 60, 80, 100, 120 and 140, or a sequence substantially similar thereto with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, 46, 62, 82, 102, 122 and 142, or a sequence substantially similar thereto with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0026] In one embodiment, the antibody or antigen-binding fragment of the antibody (a) an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 24, 40, 56, 76, 96, 116 and 136; and (b) an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 32, 48, 64, 84, 104, 124, and 144; Includes.

[0027] In related embodiments, the antibody or antigen-binding fragment of the antibody further comprises: (c) an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 20, 36, 52, 72, 92, 112 and 132; (d) an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 22, 38, 54, 74, 94, 114 and 134; (e) an LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 28, 44, 60, 80, 100, 120, and 140; and (f) an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, 46, 62, 82, 102, 122, and 142. Includes.

[0028] In one embodiment, the antibody or antigen-binding fragment thereof comprises an HCDR1 domain having an amino acid sequence selected from one of SEQ ID NOs: 4, 20, 36, 52, 72, 92, 112, and 132; an HCDR2 domain having an amino acid sequence selected from one of SEQ ID NOs: 6, 22, 38, 54, 74, 94, 114, and 134; an HCDR3 domain having an amino acid sequence selected from one of SEQ ID NOs: 8, 24, 40, 56, 76, 96, 116, and 136; and an LCVR comprising an LCDR1 domain having an amino acid sequence selected from one of SEQ ID NOs: 12, 28, 44, 60, 80, 100, 120, and 140, an LCDR2 domain having an amino acid sequence selected from one of SEQ ID NOs: 14, 30, 46, 62, 82, 102, 122, and 142, and an LCDR3 domain having an amino acid sequence selected from one of SEQ ID NOs: 16, 32, 48, 64, 84, 104, 124, and 144.

[0029] In certain embodiments, a human antibody or antigen-binding fragment of a human antibody that binds to human GCGR comprises an HCDR3 / LCDR3 amino acid sequence pair selected from the group consisting of SEQ ID NOs: 8 / 16, 24 / 32, 40 / 48, 56 / 64, 76 / 84, 86 / 88, 96 / 104, 116 / 124, and 136 / 144. Non-limiting examples of anti-GCGR antibodies having these HCDR3 / LCDR3 pairs are the antibodies designated H4H1345N, H4H1617N, H4H1765N, H4H1321B and H4H1321P, H4H1327B and H4H1327P, H4H1328B and H4H1328P, H4H1331B and H4H1331P, and H4H1339B and H4H1339P, respectively.

[0030] In one embodiment, an isolated antibody or antigen-binding fragment thereof useful in the methods provided herein that specifically binds to GCG and neutralizes at least one activity associated with GCG comprises (a) three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) amino acid sequence selected from the group consisting of SEQ ID NOs: 150, 166, 182, 198, 214, 230, 246, 262, 278, and 294; and (b) three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) amino acid sequence selected from the group consisting of SEQ ID NOs: 158, 174, 190, 206, 222, 238, 254, 270, 286, and 302.

[0031] In some embodiments, the isolated antibody or antigen-binding fragment thereof that specifically binds to GCG and neutralizes at least one activity associated with GCG comprises an HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 150, 166, 182, 198, 214, 230, 246, 262, 278, and 294, and an LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 158, 174, 190, 206, 222, 238, 254, 270, 286, and 302.

[0032] In some embodiments, the isolated antibody or antigen-binding fragment thereof that specifically binds to GCG and neutralizes at least one activity associated with GCG comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 150 / 158; 166 / 174; 182 / 190; 198 / 206; 214 / 222; 230 / 238; 246 / 254; 262 / 270; 278 / 286; and 294 / 302.

[0033] In some embodiments, the HCVR / LCVR amino acid sequence pair comprises SEQ ID NOs: 166 / 174.

[0034] In some embodiments, the HCVR / LCVR amino acid sequence pair comprises SEQ ID NO: 182 / 190.

[0035] In one embodiment, an isolated antibody or antigen-binding fragment thereof useful in accordance with the methods provided herein comprises: (a) an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 152, 168, 184, 200, 216, 232, 248, 264, 280, and 296; (b) an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 154, 170, 186, 202, 218, 234, 250, 266, 282, and 298; (c) an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 156, 172, 188, 204, 220, 236, 252, 268, 284, and 300; (d) an LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 160, 176, 192, 208, 224, 240, 256, 272, 288, and 304; (e) an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 162, 178, 194, 210, 226, 242, 258, 274, 290, and 306; and (f) an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 164, 180, 196, 212, 228, 244, 260, 276, 292, and 308. Includes.

[0036] In one embodiment, an isolated antibody or antigen-binding fragment thereof useful in accordance with the methods provided herein comprises: (a) an HCDR1 domain comprising the amino acid sequence of SEQ ID NO: 168; (b) an HCDR2 domain comprising the amino acid sequence of SEQ ID NO: 170; (c) an HCDR3 domain comprising the amino acid sequence of SEQ ID NO: 172; (d) an LCDR1 domain comprising the amino acid sequence of SEQ ID NO: 176; (e) an LCDR2 domain comprising the amino acid sequence of SEQ ID NO: 178; and (f) an LCDR3 domain comprising the amino acid sequence of SEQ ID NO: 180 Includes.

[0037] In one embodiment, an isolated antibody or antigen-binding fragment thereof useful in accordance with the methods provided herein comprises: (a) an HCDR1 domain comprising the amino acid sequence of SEQ ID NO: 184; (b) an HCDR2 domain comprising the amino acid sequence of SEQ ID NO: 186; (c) an HCDR3 domain comprising the amino acid sequence of SEQ ID NO: 188; (d) an LCDR1 domain comprising the amino acid sequence of SEQ ID NO: 192; (e) an LCDR2 domain comprising the amino acid sequence of SEQ ID NO: 194; and (f) an LCDR3 domain comprising the amino acid sequence of SEQ ID NO: 196 Includes.

[0038] Antibodies or antigen-binding fragments thereof that specifically bind to GCG, comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences provided herein, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, are also useful in accordance with the methods provided herein.

[0039] Antibodies or antigen-binding fragments thereof that specifically bind to GCG, comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences provided herein, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, are also useful in accordance with the methods provided herein.

[0040] Antibodies or antigen-binding fragments thereof that specifically bind to GCG, comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences provided herein, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, are also useful in accordance with the methods provided herein.

[0041] Antibodies or antigen-binding fragments thereof that specifically bind to GCG, comprising a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences provided herein, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, are also useful in accordance with the methods provided herein.

[0042] Antibodies or antigen-binding fragments thereof that specifically bind to GCG, comprising a light chain CDR2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences provided herein, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, are also useful in accordance with the methods provided herein.

[0043] Antibodies or antigen-binding fragments thereof that specifically bind to GCG, comprising a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed herein, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, are also useful in accordance with the methods provided herein.

[0044] Antibodies or antigen-binding fragments thereof that specifically bind to GCG, comprising an HCDR3 and an LCDR3 amino acid sequence pair (HCDR3 / LCDR3) comprising any of the HCDR3 amino acids provided herein paired with any of the LCDR3 amino acid sequences provided herein, are also useful in accordance with the methods provided herein. According to certain embodiments, the antibody or antigen-binding fragment thereof comprises an HCDR3 / LCDR3 amino acid sequence pair contained in any of the exemplary anti-GCG antibodies provided herein. In certain embodiments, the HCDR3 / LCDR3 amino acid sequence pair comprises SEQ ID NOs: 172 / 180.

[0045] Antibodies or antigen-binding fragments thereof that specifically bind to GCG, comprising the set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in any of the exemplary anti-GCG antibodies provided herein, are also useful according to the methods provided herein. In certain embodiments, the set of HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 amino acid sequences comprises SEQ ID NOs: 168 / 170 / 172 / 176 / 178 / 180. In certain embodiments, the set of HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 amino acid sequences comprises SEQ ID NOs: 184 / 186 / 188 / 192 / 194 / 196.

[0046] In related embodiments, an antibody or antigen-binding fragment thereof that specifically binds to GCG comprises a set of six CDRs (i.e., HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3) contained within the HCVR / LCVR amino acid sequence pair defined by any of the exemplary anti-GCG antibodies provided herein. For example, an antibody or antigen-binding fragment thereof that specifically binds to GCG comprises a set of HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 amino acid sequences contained within the HCVR / LCVR amino acid sequence pair selected from the group consisting of 166 / 174; 182 / 190; 198 / 206; 214 / 222; 230 / 238; 246 / 254; 262 / 270; 278 / 286 and 294 / 302.

[0047] Non-limiting examples of antibodies that specifically bind to GCG and comprise the CDR sequences provided above include HIH059P, H4H10223P, H4H10231P, H4H10232P, H4H10236P, H4H10237P, H4H10238P, H4H10250P, H4H10256P, and H4H10270P.

[0048] Methods and techniques for identifying CDRs in HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs in the specific HCVR and / or LCVR amino acid sequences disclosed herein.Exemplary conventions that can be used to identify the boundaries of CDRs include, for example, Kabat definition, Chothia definition, and AbM definition.In general terms, Kabat definition is based on sequence variability, Chothia definition is based on the position of structural loop regions, and AbM definition is a compromise between Kabat and Chothia methods.See, for example, Kabat, (1991) "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md.; Al-Lazikani et al., (1997) J. Mol. Biol. 273:927-948, and Martin et al., (1989) Proc. Natl. Acad. Sci. USA 86:9268-9272. Public databases are also available for identifying CDR sequences within antibodies.

[0049] In some embodiments, the subject with hyperammonemia may suffer from one of the following conditions or diseases: liver diseases and conditions associated with liver disease, such as urea cycle disorders, hepatic encephalopathy, and hepatic halitosis, and conditions or diseases associated with urea cycle disorders or with the presence of enzyme deficiencies reported to cause hyperammonemia. In some embodiments, elevated ammonia levels are detected in the subject's serum. In some embodiments, elevated glutamine levels are detected in the subject's serum.

[0050] In some embodiments, the enzyme deficiency associated with a urea cycle disorder is selected from carbamyl phosphate synthetase (CPS1), N-acetylglutamate synthetase (NAGS), ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL), and arginase (AR1). In some embodiments, the urea cycle disorder is associated with a transporter deficiency selected from ornithine translocase (ORNT1; ornithine / citrulline carrier; solute carrier family 25, member 15) and citrine (aspartate / glutamate transporter; solute carrier family 25, member 13).

[0051] In some embodiments, the glucagon signaling pathway antagonist is administered in an amino acid formulation, such as one selected from Cyclinex (e.g., Cyclinex-1 or -2), EAAs (essential amino acids), UCD I or II (urea cycle disorder-1 or -II), and individual branched chain amino acids.

[0052] In some aspects, the glucagon signaling pathway antagonist is administered with an antioxidant or electrolyte.

[0053] In some embodiments, the glucagon signaling pathway antagonist is administered with L-citrulline or L-arginine free base.

[0054] In some aspects, the glucagon signaling pathway antagonist is administered in conjunction with hemodialysis or continuous renal replacement.

[0055] In some aspects, a composition comprising a glucagon signaling pathway antagonist is administered to a subject in combination with at least one additional therapeutic agent. The additional therapeutic agent can be any agent that alleviates or reduces symptoms and signs associated with hyperammonemia and / or a urea cycle disorder. In some embodiments, the at least one additional therapeutic agent is selected from the following: a non-absorbable antibiotic (rifaximin or lactulose), sodium phenylbutyrate, sodium benzoate, sodium phenylacetate, glycerol phenylbutyrate, carbamyl glutamate (Carbaglu®), a second GCG inhibitor, and a second GCGR antagonist.

[0056] Provided herein are methods for reducing the amount and / or dose of sodium phenylbutyrate or sodium benzoate required to treat a subject with hyperammonemia. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a composition comprising a glucagon signaling pathway antagonist. In some aspects, the glucagon signaling pathway antagonist is administered in combination with sodium phenylbutyrate or sodium benzoate.

[0057] [The present invention 1001] 1. A method for treating a condition or disease associated with or characterized in part by hyperammonemia, or at least one symptom or complication associated with said condition or disease, in a subject, comprising: administering to the subject a therapeutically effective amount of a composition comprising a glucagon signaling pathway antagonist; Decreasing serum ammonia levels, or reduce the effects of said condition or disease; or At least one symptom or complication associated with said condition or disease is alleviated or reduced in severity The method comprises administering the compound as described above. [The present invention 1002] 1001. The method of claim 1001, wherein said hyperammonemia is acquired. [The present invention 1003] 1001. The method of claim 10, wherein said hyperammonemia is congenital hyperammonemia. [The present invention 1004] The congenital hyperammonemia is (i) a deficiency in one or more urea cycle enzymes selected from the group consisting of carbamyl phosphate synthetase (CPS1), N-acetylglutamate synthetase (NAGS), ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL), and arginase (AR1); (ii) a defect in one or more urea cycle transporters selected from ornithine translocase (ORNT1) and citrin; (iii) methylmalonic aciduria, propionic aciduria, and / or isovaleric aciduria; (iv) medium-chain acyl-CoA dehydrogenase deficiency, multiple acyl-CoA dehydrogenase deficiency, carnitine palmitoyltransferase II deficiency, carnitine-acylcarnitine translocase, lysinuric protein intolerance, pyrroline-5-carboxylic acid synthetase deficiency, pyruvate carboxylase deficiency, ornithine aminotransferase deficiency, carbonic anhydrase Va deficiency, hyperinsulinemia-hyperammonemia syndrome, mitochondrial disorders, and / or glutamine synthetase deficiency; (v) acute or chronic liver failure and its complications; (vi) treatment with the therapeutic agents L-asparaginase, pegaspargase, 5-pentanoic acid, valproic acid, corticosteroids, and / or cyclophosphamide; (vii) herpes simplex infection, hepatitis B infection, and / or infection with urease-producing organisms; and / or (viii) Transient hyperammonemia in subjects on total parenteral nutrition, L-asparaginase treatment, nutritional carnitine deficiency, cystoscopy with glycine-containing solutions, after lung / bone marrow transplantation, congenital anomalies of blood vessels, and / or who are neonates with relative arginine deficiency Any of the methods 1001 to 1003 of the present invention, which is caused by the above. [The present invention 1005] The method of any of claims 1001 to 1004, wherein said glucagon signaling pathway antagonist is a glucagon (GCG) inhibitor or a glucagon receptor (GCGR) antagonist. [The present invention 1006] The GCG inhibitor or GCGR antagonist is (i) one or more selected from the group consisting of antisense molecules, anti-GCGR antibodies, small molecule inhibitors, shRNA, siRNA, peptide inhibitors, DARPins, spiegelmers, aptamers, engineered Fn3-type domains, anti-GCG antibodies, and derivatives thereof; (ii) an isolated human monoclonal antibody or antigen-binding fragment thereof; (iii) a complementarity determining region (CDR) of a heavy chain variable region (HCVR), wherein the HCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 70, 86, 90, 106, 110, 126, 130, and 146; and / or a complementarity determining region (CDR) of a light chain variable region (LCVR), wherein the LCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 68, 78, 88, 98, 108, 118, 128, 138, and 148. a GCGR antagonist, which is an isolated antibody or antigen-binding fragment thereof comprising: (iv)(a) an HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 70, 86, 90, 106, 110, 126, 130, and 146; and / or (b) an LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 68, 78, 88, 98, 108, 118, 128, 138, and 148 a GCGR antagonist, which is an isolated antibody or antigen-binding fragment thereof comprising: (v) a GCGR antagonist that is an isolated antibody or antigen-binding fragment thereof comprising a HCVR / LCVR sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 68, 70 / 78, 86 / 88, 90 / 98, 106 / 108, 110 / 118, 126 / 128, 130 / 138, and 146 / 148; (vi)(a) three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) amino acid sequence selected from the group consisting of SEQ ID NOs: 150, 166, 182, 198, 214, 230, 246, 262, 278, and 294; and / or (b) three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) amino acid sequence selected from the group consisting of SEQ ID NOs: 158, 174, 190, 206, 222, 238, 254, 270, 286, and 302; a GCG inhibitor, which is an isolated antibody or antigen-binding fragment thereof comprising: (vii) an HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 150, 166, 182, 198, 214, 230, 246, 262, 278, and 294, and / or an LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 158, 174, 190, 206, 222, 238, 254, 270, 286, and 302. and / or a GCG inhibitor, which is an isolated antibody or antigen-binding fragment thereof comprising: (viii) a GCG inhibitor, which is an isolated antibody or antigen-binding fragment thereof comprising an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 150 / 158, 166 / 174, 182 / 190, 198 / 206, 214 / 222, 230 / 238, 246 / 254, 262 / 270, 278 / 286, and 294 / 302. The method of the present invention 1005. [The present invention 1007] The method of any of claims 1001 to 1006, wherein said composition is administered to said subject in combination with at least one additional therapeutic agent or supplement. [The present invention 1008] The glucagon signaling pathway antagonist is (i) one or more amino acid formulations selected from Cyclinex, EAA, UCD-I, UCD-II, and individual branched-chain amino acids; (ii) antioxidants and / or electrolytes; (iii) L-citrulline and / or L-arginine free base; (iv) hemodialysis and / or continuous renal replacement; and / or (v) non-absorbable antibiotics, rifaximin, lactulose, sodium phenylbutyrate, sodium benzoate, sodium phenylacetate, glycerol phenylbutyrate, carbamyl glutamate, a second GCG inhibitor, and / or a second GCGR antagonist The method of any one of claims 1001 to 1007, wherein the method is administered in combination with [The present invention 1009] A method for treating a subject having a urea cycle disorder, wherein the subject exhibits elevated levels of ammonia, the method comprising administering to the subject a therapeutically effective amount of a composition comprising a glucagon signaling pathway antagonist. [The present invention 1010] the subject having a urea cycle disorder (i) one or more deficiencies in urea cycle enzymes selected from the group consisting of carbamyl phosphate synthetase (CPS1), N-acetylglutamate synthetase (NAGS), ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL), and arginase (AR1); and / or (ii) one or more defects in urea cycle transporters selected from ornithine translocase (ORNT1) and citrin The method of the present invention 1009, wherein the patient is suffering from [The present invention 1011] The GCG inhibitor or GCGR antagonist is (i) one or more selected from the group consisting of antisense molecules, anti-GCGR antibodies, small molecule inhibitors, shRNA, siRNA, peptide inhibitors, DARPins, spiegelmers, aptamers, engineered Fn3-type domains, anti-GCG antibodies, and derivatives thereof; (ii) an isolated human monoclonal antibody or antigen-binding fragment thereof; (iii) a CDR of an HCVR, wherein the HCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 70, 86, 90, 106, 110, 126, 130, and 146, and / or a CDR of an LCVR, wherein the LCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 68, 78, 88, 98, 108, 118, 128, 138, and 148. a GCGR antagonist, which is an isolated antibody or antigen-binding fragment thereof comprising: (iv)(a) an HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 70, 86, 90, 106, 110, 126, 130, and 146; and / or (b) an LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 68, 78, 88, 98, 108, 118, 128, 138, and 148 a GCGR antagonist, which is an isolated antibody or antigen-binding fragment thereof comprising: (v) a GCGR antagonist that is an isolated antibody or antigen-binding fragment comprising a HCVR / LCVR sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 68, 70 / 78, 86 / 88, 90 / 98, 106 / 108, 110 / 118, 126 / 128, 130 / 138, and 146 / 148; (vi)(a) three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) amino acid sequence selected from the group consisting of SEQ ID NOs: 150, 166, 182, 198, 214, 230, 246, 262, 278, and 294; and / or (b) three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) amino acid sequence selected from the group consisting of SEQ ID NOs: 158, 174, 190, 206, 222, 238, 254, 270, 286, and 302; a GCG inhibitor, which is an isolated human monoclonal antibody or an antigen-binding fragment thereof comprising: (vii) an HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 150, 166, 182, 198, 214, 230, 246, 262, 278, and 294, and / or an LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 158, 174, 190, 206, 222, 238, 254, 270, 286, and 302. and / or a GCG inhibitor, which is an isolated antibody or antigen-binding fragment thereof comprising: (viii) A GCG inhibitor, which is an isolated antibody or antigen-binding fragment thereof comprising an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 150 / 158, 166 / 174, 182 / 190, 198 / 206, 214 / 222, 230 / 238, 246 / 254, 262 / 270, 278 / 286, and 294 / 302. The method of any one of 1009 to 1010 of the present invention. [The present invention 1012] The method of any of claims 1009 to 1011, wherein the composition is administered to the subject in combination with at least one additional therapeutic agent or supplement. [The present invention 1013] The glucagon signaling pathway antagonist is (i) one or more amino acid formulations selected from Cyclinex, EAA, UCD-I, UCD-II, and individual branched-chain amino acids; (ii) antioxidants and / or electrolytes; (iii) L-citrulline and / or L-arginine free base; (iv) hemodialysis and / or continuous renal replacement; and / or (v) non-absorbable antibiotics, rifaximin, lactulose, sodium phenylbutyrate, sodium benzoate, sodium phenylacetate, glycerol phenylbutyrate, carbamyl glutamate, a second GCG inhibitor, and / or a second GCGR antagonist The method of any one of claims 1009 to 1012, wherein the method is administered in combination with [The present invention 1014] A method for reducing the amount and / or dose of sodium phenylbutyrate or sodium benzoate required to treat a subject having hyperammonemia, comprising administering to the subject a therapeutically effective amount of a composition comprising a glucagon signaling pathway antagonist. [The present invention 1015] The method of claim 1014, wherein said glucagon signaling pathway antagonist is administered in combination with sodium phenylbutyrate and / or sodium benzoate. [The present invention 1016] The method of any one of claims 1014 to 1015, wherein the glucagon signaling pathway antagonist is a GCGR antagonist and / or a GCG inhibitor. [The present invention 1017] The glucagon signaling pathway antagonist is (i) one or more selected from the group consisting of antisense molecules, anti-GCGR antibodies, small molecule inhibitors, shRNA, siRNA, peptide inhibitors, DARPins, spiegelmers, aptamers, engineered Fn3-type domains, anti-GCG antibodies, and derivatives thereof; (ii) an isolated human monoclonal antibody or antigen-binding fragment thereof; (iii) a CDR of an HCVR, wherein the HCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 70, 86, 90, 106, 110, 126, 130, and 146, and / or a CDR of an LCVR, wherein the LCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 68, 78, 88, 98, 108, 118, 128, 138, and 148. a GCGR antagonist, which is an isolated antibody or antigen-binding fragment thereof comprising: (iv)(a) an HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 70, 86, 90, 106, 110, 126, 130, and 146; and / or (b) an LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 68, 78, 88, 98, 108, 118, 128, 138, and 148 a GCGR antagonist, which is an isolated antibody or antigen-binding fragment thereof comprising: (v) a GCGR antagonist that is an isolated antibody or antigen-binding fragment comprising a HCVR / LCVR sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 68, 70 / 78, 86 / 88, 90 / 98, 106 / 108, 110 / 118, 126 / 128, 130 / 138, and 146 / 148; (vi)(a) three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) amino acid sequence selected from the group consisting of SEQ ID NOs: 150, 166, 182, 198, 214, 230, 246, 262, 278, and 294; and / or (b) three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) amino acid sequence selected from the group consisting of SEQ ID NOs: 158, 174, 190, 206, 222, 238, 254, 270, 286, and 302; a GCG inhibitor, which is an isolated antibody or antigen-binding fragment thereof comprising: (vii) an HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 150, 166, 182, 198, 214, 230, 246, 262, 278, and 294, and / or an LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 158, 174, 190, 206, 222, 238, 254, 270, 286, and 302. and / or a GCG inhibitor, which is an isolated antibody or antigen-binding fragment thereof comprising: (viii) A GCG inhibitor, which is an isolated antibody or antigen-binding fragment thereof comprising an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 150 / 158, 166 / 174, 182 / 190, 198 / 206, 214 / 222, 230 / 238, 246 / 254, 262 / 270, 278 / 286, and 294 / 302. Any of the methods of present invention 1014 to 1016, [The present invention 1018] 1. A method for reducing blood ammonia levels or for treating a condition or disease associated with or characterized in part by hyperammonemia, or at least one symptom or complication associated with said condition or disease, in a subject, comprising: administering to the subject a therapeutically effective amount of a composition comprising a glucagon signaling pathway antagonist such that blood ammonia levels are lowered, or the effects of the condition or disease are reduced, or at least one symptom or complication associated with the condition or disease is alleviated or reduced in severity. A method comprising: [The present invention 1019] The glucagon signaling pathway antagonist is (i) one or more selected from the group consisting of antisense molecules, anti-GCGR antibodies, small molecule inhibitors, shRNA, siRNA, peptide inhibitors, DARPins, spiegelmers, aptamers, engineered Fn3-type domains, anti-GCG antibodies, and derivatives thereof; (ii) an isolated human monoclonal antibody or antigen-binding fragment thereof; (iii) a CDR of an HCVR, wherein the HCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 70, 86, 90, 106, 110, 126, 130, and 146, and / or a CDR of an LCVR, wherein the LCVR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 68, 78, 88, 98, 108, 118, 128, 138, and 148. a GCGR antagonist, which is an isolated antibody or antigen-binding fragment thereof comprising: (iv)(a) an HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 70, 86, 90, 106, 110, 126, 130, and 146; and / or (b) an LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 68, 78, 88, 98, 108, 118, 128, 138, and 148 a GCGR antagonist, which is an isolated antibody or antigen-binding fragment thereof comprising: (v) a GCGR antagonist that is an isolated antibody or antigen-binding fragment comprising a HCVR / LCVR sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 68, 70 / 78, 86 / 88, 90 / 98, 106 / 108, 110 / 118, 126 / 128, 130 / 138, and 146 / 148; (vi)(a) three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) amino acid sequence selected from the group consisting of SEQ ID NOs: 150, 166, 182, 198, 214, 230, 246, 262, 278, and 294; and / or (b) three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) amino acid sequence selected from the group consisting of SEQ ID NOs: 158, 174, 190, 206, 222, 238, 254, 270, 286, and 302; a GCG inhibitor, which is an isolated antibody or antigen-binding fragment thereof comprising: (vii) an HCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 150, 166, 182, 198, 214, 230, 246, 262, 278, and 294, and / or an LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 158, 174, 190, 206, 222, 238, 254, 270, 286, and 302. and / or a GCG inhibitor, which is an isolated antibody or antigen-binding fragment thereof comprising: (viii) A GCG inhibitor, which is an isolated antibody or antigen-binding fragment thereof comprising an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 150 / 158, 166 / 174, 182 / 190, 198 / 206, 214 / 222, 230 / 238, 246 / 254, 262 / 270, 278 / 286, and 294 / 302. The method of the present invention 1018. [The present invention 1020] A method for reducing mortality, alleviating excessive weight loss, and / or lowering blood glucose in a subject having a urea cycle disorder, comprising administering to the subject a therapeutically effective amount of a composition comprising a glucagon signaling pathway antagonist. [The present invention 1021] The method of claim 1020, wherein the subject is on a high protein diet. Other objects and advantages will become apparent from consideration of the following detailed description. [Brief explanation of the drawings]

[0058] [Figure 1] FIG. 1 is a graphical representation of Table 3, showing the mean±SEM of non-fasting blood glucose levels for the four groups of mice. [Figure 2] FIG. 2 is a graphical representation of Table 4, showing the mean±SEM change in body weight from baseline for the four groups of mice. [Figure 3] Figure 3 is a graphical representation of Table 5 and shows the mean ± SEM plasma ammonia levels for the four groups of mice at baseline and at weeks 2, 3, 4, 6, 7, and 8. [Figure 4] FIG. 4 shows the survival curves of the four groups of mice. [Figure 5]Figure 5 shows plasma ammonia levels over time in wild-type and Otc mutant mice on a 30% protein diet treated with an isotype control or the H4H1327P antibody. **: p<0.01 between the two treatments in Otc mutant mice, ***: p<0.001 between the two treatments in Otc mutant mice, ****: p<0.0001 between the two treatments in Otc mutant mice. [Figure 6] FIG. 6 shows survival curves for wild-type and Otc mutant mice on a 30% protein diet treated with isotype control or H4H1327P antibody. [Figure 7] Figure 7 shows the change in body weight over time from baseline in wild-type and Otc mutant mice on a 30% protein diet treated with isotype control or H4H1327P antibody. *: p<0.05 between the two treatments in Otc mutant mice, ****: p<0.0001 between the two treatments in Otc mutant mice. [Figure 8] Figure 8 shows blood glucose levels (mg / dL) over time in wild-type and Otc mutant mice on a 30% protein diet treated with an isotype control or the H4H1327P antibody. *: p<0.05 between the two treatments in Otc mutant mice, ***: p<0.001 between the two treatments in Otc mutant mice, ****: p<0.0001 between the two treatments in Otc mutant mice, ^^^^: p<0.0001 between the two treatments in wild-type mice. DETAILED DESCRIPTION OF THE INVENTION

[0059] explanation Before describing the present method, it is to be understood that the invention is not limited to the particular methods and experimental conditions described herein, as such methods and conditions may vary. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0060] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more methods, and / or steps of the type described herein and / or that will become apparent to those skilled in the art upon reading this disclosure and so forth.

[0061] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All patents, patent applications, and non-patent publications mentioned herein are incorporated by reference in their entirety.

[0062] overview Amino acid metabolic enzymes are involved in ammonia production through the breakdown of amino acids. These enzymes include cystathionase (CTH), serine dehydratase (SDS), ornithine aminotransferase (OAT), and glutaminase 2 (GLS-2). Ammonia derived from amino acid metabolism, as well as ammonia absorbed from the intestine, produced by skeletal muscle during exercise, or generated by ammonium production by the kidney, is converted to urea by the urea cycle in hepatocytes. The urea cycle is the primary mechanism for the clearance of ammonia resulting from protein turnover and for the metabolism of other nitrogenous metabolic compounds, such as adenosine monophosphate, by converting ammonia to urea. Urea is then excreted in the urine. Glutamine synthetase activity is also required to reduce plasma ammonia levels. Glucagon signaling pathway inhibitors, such as GCGR antibodies, target amino acid transporters to reduce amino acid uptake into hepatocytes.

[0063] Urea cycle disorders result from genetic mutations in genes encoding one of the enzymes involved in the conversion of ammonia to urea. Disruption of the urea cycle, typically through urea cycle enzyme or transporter deficiencies, through metabolite accumulation, or substrate deficiency, leads to the accumulation of nitrogen in the form of ammonia, a condition called hyperammonemia. Elevated blood ammonia can cause irreversible brain damage, coma, and death. Seizures are common in acute hyperammonemia and may result from brain injury. Subclinical seizures are common during acute hyperammonemic episodes, especially in newborns, and can occur during elevated glutamine levels even before ammonia levels reach their peak. Ammonia can cause brain injury through various mechanisms, a major component of which is cerebral edema through increased glutamine, although the specific roles of ammonia, glutamate, and glutamine in cerebral edema remain unclear. Furthermore, adverse effects on the nitric oxide production system, possibly due to arginine deficiency resulting from ASL deficiency, may also contribute to the injury.

[0064] In addition to increased plasma ammonia levels, the following changes in plasma and / or urine levels may be seen in one or more forms of urea cycle disorders: Increase or decrease in plasma citrulline; Increased plasma and / or urinary levels of argininosuccinic acid (ASA); Increased or decreased plasma arginine; Increased plasma ornithine; Increased plasma glutamine; Increased plasma alanine; Increased plasma asparagine; Increased urinary orotic acid; or Increased urinary homocitrulline.

[0065] In some subjects, urea cycle disorders are associated with inborn errors of urea synthesis or the presence of urea cycle enzyme deficiencies: carbamyl phosphate synthetase (CPS1), N-acetylglutamate synthetase (NAGS), ornithine transcarbamylase (OTC deficiency), argininosuccinate synthetase (ASS, citrullinemia), argininosuccinate lyase (AL or ASA lyase, argininosuccinic aciduria), and arginase (AR1).

[0066] Other inborn errors of metabolism can also cause hyperammonemia: ornithine aminotransferase (OAT) deficiency (in newborns), tyrosinemia type 1, galactosemia, citrin deficiency leading to mitochondrial disorders, citrullinemia type 2 (CTLN2), and neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD). CTLN2 is a late-onset disorder in which there is no liver dysfunction, but subjects experience recurrent hyperammonemia, delirium, irritability, and fatty liver infiltration. Citrin deficiency results from mutations in the gene SLC25A13. Because citrin functions to transport aspartate / glutamate across the mitochondrial membrane to the cytoplasm, citrin deficiency results in a decrease in cytosolic aspartate, which limits the activity of the enzyme argininosuccinate synthase. Dysfunction of another transporter, ornithine translocase, causes hyperornithineemia, homocitrullinuia, and hyperammonemia, as reduced ornithine transport into mitochondria results in orotic aciduria and deficiency in urea synthesis.

[0067] Other congenital disorders that can cause hyperammonemia include propionic acidemia, isolated methylmalonic acidemia, isovaleric acidemia, carbonic anhydrase Va deficiency, lysinuric protein intolerance, carnitine palmitoyltransferase II deficiency, carnitine-acylcarnitine translocase, pyrroline-5-carboxylate synthetase deficiency, pyruvate carboxylase deficiency, carbonic anhydrase Va deficiency, hyperinsulinemia-hyperammonemia syndrome, mitochondrial disorders, glutamine synthetase deficiency, fatty acid oxidation disorders (e.g., short-chain acyl-CoA dehydrogenase deficiency, medium-chain acyl-CoA dehydrogenase deficiency, multiple acyl-CoA dehydrogenase deficiency, very-long-chain acyl-CoA dehydrogenase deficiency), and hyperinsulinemia-hyperammonemia syndrome (familial hyperinsulinism). See Haberle, Archives of Biochemistry and Biophysics, 536:101-108, 2013.

[0068] The onset and severity of urea cycle disorders are highly variable, depending on the degree of enzyme or transporter dysfunction. Severe deficiency or total lack of activity of any of the first four enzymes in the pathway (CPS1, OTC, ASS1, and ASL) or the cofactor producer (NAGS) leads to the accumulation of ammonia and other precursor metabolites during the first few days of life. Newborns with severe urea cycle dysfunction develop devastating illness within the first few days of life. Symptoms include irritability, poor feeding, vomiting, and lethargy, followed by seizures, hypotonia, respiratory distress, and coma.

[0069] Individuals with partial urea cycle dysfunction, including children and adults, have insidious symptoms, and diagnosis is often difficult given that the symptoms are generally unrecognized. Children with mild or moderate urea cycle dysfunction may have early symptoms including failure to thrive, inconsolable crying, agitated or hyperactive behavior, and aversion to high-protein foods. Later symptoms include frequent vomiting, lethargy, delirium, and, if untreated, hyperammonemic coma and death.

[0070] Late-onset disease in adults is identified when acute onset of hyperammonemia is precipitated by metabolic stressors, including viral infection, excessive exercise or dieting, postpartum stress, high-protein parenteral nutrition, gastrointestinal bleeding, valproic acid administration, prednisone or other corticosteroid administration, infection, and postoperative stress. Symptoms typically include disorientation, confusion, slurred speech, combativeness or agitation, stroke-like symptoms, lethargy, and episodes of delirium. Subjects are often treated for psychiatric symptoms before diagnosis. Without treatment, subjects who experience acute onset of hyperammonemia are at risk for permanent brain damage, coma, and death.

[0071] An exemplary delayed-onset subject may be a bodybuilder who eats a high-protein diet. Such subjects are at high risk for undiagnosed urea cycle disorders, which, when combined with a high-protein diet, can cause acute onset of hyperammonemia. Such subjects may be inappropriately diagnosed because they are otherwise considered to be very healthy individuals. Delayed diagnosis can lead to coma and death.

[0072] Additional causes of hyperammonemia, i.e., acquired hyperammonemia, include, for example, hepatic encephalopathy in subjects with advanced liver disease, hepatic halitosis (a late sign of liver failure), hepatic vascular bypass, and liver disease and its complications such as biliary atresia, administration of toxic levels of valproic acid (a metabolite of NAGS that inhibits and also depletes carnitine), corticosteroids, or cyclophosphamide, herpes simplex infection, and gastrointestinal bacterial overgrowth. Hyperammonemia can also be caused by infection with urease-producing organisms (increased ammonia production in the intestine or urinary tract).

[0073] Additionally, hyperammonemia can be caused by total parenteral nutrition (which has a relative arginine deficiency), treatment with L-asparaginase (increased ammonia production due to asparagine hydrolysis), nutritional carnitine deficiency (impaired fatty acid oxidation leading to acetyl-CoA deficiency), cystoscopy with glycine-containing solutions (increased ammonia production resulting from nitrogen overload), after lung / bone marrow transplantation (which reduces glutamine synthetase activity), congenital vascular anomalies, or transient hyperammonemia in newborns (see Haberle, Archives of Biochemistry and Biophysics, 536:101–108, 2013).

[0074] Treatment options initially include hemodialysis or continuous renal replacement therapy (RRT) as soon as it becomes clear that the subject is experiencing an acute episode of hyperammonemia. Hemodialysis or RRT may be stopped if plasma ammonia levels fall below 80 μmol / L or below 120 μmol / L. In addition, oral or parenteral protein administration is discontinued, while administration of calories from glucose and fat helps prevent excessive catabolic states. Once a diagnosis of UCD is made, treatment for acute episodes can be initiated and tailored to the specific urea cycle disorder. Treatments such as intravenous or oral administration of sodium phenylacetate, sodium benzoate, sodium phenylbutyrate, or glycerol phenylbutyrate remove ammonia and redirect nitrogen from the urea cycle for excretion through the kidneys. Sodium phenylacetate combines with glutamine to produce phenylacetylglutamine, which is excreted by the kidneys. Sodium benzoate conjugates with glycine to produce sodium hippurate, which is also excreted by the kidneys. Unfortunately, for subjects with recurrent hyperammonemia or who are refractory to conventional treatment, liver transplantation is the best treatment option.

[0075] While not wishing to be held by theory, antagonists of GCGR have been found to reduce the expression of amino acid metabolic enzymes in the liver of mice and monkeys, thus providing a method for treating hyperammonemia and urea cycle disorders by reducing the amount of ammonia entering the urea cycle by blocking the glucagon signaling pathway.

[0076] To date, no studies have examined the effect of antagonizing the glucagon signaling pathway on urea cycle disorders or conditions or diseases associated with hyperammonemia. The studies described in the Examples use antagonists of GCGR as exemplary inhibitors of the glucagon signaling pathway in mouse models of urea cycle disorders and demonstrate the effect on hyperammonemia and death over several weeks of treatment.

[0077] definition The "glucagon receptor," also known as "GCGR," belongs to the G protein-coupled receptor class 2 family and consists of a long amino-terminal extracellular domain, seven transmembrane segments, and an intracellular C-terminal domain. Glucagon receptors are specifically expressed on the surface of hepatocytes, where they bind to glucagon and transduce the signal provided thereby into the cells. Therefore, the term "glucagon receptor" also refers to one or more receptors that specifically interact with glucagon to produce a biological signal. DNA sequences encoding rat and human glucagon receptors have been isolated and disclosed in the art (EP 0658200 B1). Homologues from mice and cynomolgus monkeys have also been isolated and sequenced (Burcelin et al., (1995) Gene 164:305-310; McNally et al., (2004) Peptides 25:1171-1178). As used herein, "glucagon receptor" and "GCGR" are used interchangeably. As used herein, the terms "GCGR," "hGCGR," or fragments thereof refer to the human GCGR protein or fragments thereof, unless specified as being from a non-human species, e.g., "mouse GCGR," "rat GCGR," or "monkey GCGR."

[0078] The term "GCGR antagonist" refers to an inhibitor, antagonist, or inverse agonist of the GCGR signaling pathway. A "GCG inhibitor" may prevent glucagon from binding to the receptor. A GCGR inhibitor may also prevent glucagon from binding to the receptor. However, both may effectively block or attenuate receptor activation or interfere with the signaling cascade downstream of GCGR activation, and are collectively referred to as "glucagon signaling pathway antagonists."

[0079] The term "inhibitor" or "antagonist" includes substances that slow or block a chemical or physiological reaction or response, for example, glucagon signaling pathway antagonists.

[0080] GCGR antagonists can bind to glucagon receptors, thereby antagonizing the activity of GCG mediated by GCGR.By antagonizing the binding and activity of GCG at GCGR, inhibiting the activity of GCG reduces the expression of enzymes involved in amino acid metabolism.Methods for determining the binding of potential antagonists with glucagon receptors are known in the art, and means for determining interference with glucagon activity at glucagon receptors are generally published.For example, see SE de Laszlo et al., (1999) Bioorg.Med.Chem.Lett.9:641-646.

[0081] The glucagon signaling pathway antagonist may be useful herein, or may be a GCG inhibitor that has a small molecule compound, or in other words, a low molecular weight organic compound, as its functional component.Small molecules are typically less than 800 daltons.Furthermore, CRISPR technology can be used to knock down GCG or GCGR expression.Thus, in some embodiments, the glucagon signaling pathway antagonist may be selected from small molecule inhibitors, shRNA, siRNA, peptide inhibitors, CRISPR technology (Clustered regularly interspaced short palindromic repeats, CRISPR technology can generate GCGR knockdown or deletion of regulatory sequences that affect GCGR activity), antisense inhibitors, DARPin, spiegelmers, aptamers, engineered Fn3-type domains, GCG or GCGR neutralizing monoclonal antibodies, and their derivatives.

[0082] Examples of glucagon signaling pathway antagonists include, but are not limited to, antibodies (human or humanized) to GCG or GCGR, or antigen-binding portions thereof, that block or inhibit the activity of the GCGR signaling pathway. Exemplary GCGR antagonists that can be used in the methods described herein include an isolated human monoclonal antibody or antigen-binding fragment thereof comprising (a) an HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 70, 86, 90, 106, 110, 126, 130, and 146, and / or (b) an LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 68, 78, 88, 98, 108, 118, 128, 138, and 148. Exemplary GCG inhibitors that can be used in the methods described herein include an isolated human monoclonal antibody or antigen-binding fragment thereof comprising: (a) an HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 150, 166, 182, 198, 214, 230, 246, 262, 278, and 294; and / or (b) an LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 158, 174, 190, 206, 222, 238, 254, 270, 286, and 302.

[0083] A "therapeutically effective dose" is the dose for which it is administered that produces the desired effect. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0084] The phrase "substantially identical" refers to a protein sequence that has at least 95% identity to (a) an HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 70, 86, 90, 106, 110, 126, 130, and 146, and / or (b) an LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 68, 78, 88, 98, 108, 118, 128, 138, and 148, and that is capable of binding to GCGR and inhibiting the biological activity of GCGR. The phrase "substantially identical" refers to a protein sequence that has at least 95% identity to (a) an HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 150, 166, 182, 198, 214, 230, 246, 262, 278, and 294, and / or (b) an LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 158, 174, 190, 206, 222, 238, 254, 270, 286, and 302, and that is capable of binding to GCG and inhibiting the biological activity of GCG.

[0085] The term "identity" or "homology" is understood to mean the percentage of amino acid residues in a candidate sequence that are identical to the residues of the corresponding sequence being compared, after aligning the sequences and introducing gaps, as needed to achieve the maximum percent identity across the entire sequence, and without considering any conservative substitutions as part of the sequence identity. Neither N- nor C-terminal extensions nor insertions are construed as reducing identity or homology. Methods and computer programs for alignment are well known in the art. Sequence identity may be measured using sequence analysis software (e.g., Sequence Analysis Software Package, Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Ave., Madison, Wis. 53705). This software matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications.

[0086] The term "treat" (or "treating" or "treatment") refers to a process that includes slowing, interrupting, inhibiting, preventing, suppressing, halting, reducing, ameliorating, or reversing the progression, duration, or severity of an existing symptom, disorder, condition, or disease, but does not necessarily include the total elimination of all disease-related symptoms, conditions, or disorders through the use of the GCG inhibitors or GCGR antagonists described herein. Furthermore, "treat," "treatment," or "treating" refers to an approach to obtaining beneficial or desired results, including clinical results including, but not limited to, one or more of the following: inhibiting, slowing, or preventing the progression of hyperammonemia and / or urinary cycle disorders; inhibiting, slowing, or preventing the progression of a hyperammonemia-associated disease, or a disease characterized by elevated plasma ammonia levels, such as in chronic liver disease or toxic administration of valproic acid, or a condition or disease associated with the presence of a genetic variant reported to cause a urea cycle disorder; or inhibiting, preventing, or ameliorating at least one symptom associated with a hyperammonemia-associated disease; or lowering blood ammonia levels so as to reduce the impact of a hyperammonemia-associated condition or disease or to alleviate or reduce in severity at least one symptom or complication associated with the condition or disease. "Treatment" or "treating," as used herein, also refers to reducing the dose of other medications needed to treat the disease and / or prolong the subject's life, and increasing the quality of life of a subject suffering from a disease. For example, "treatment" or "treating" can include reducing the amount and / or dosage of sodium phenylbutyrate or sodium benzoate required to treat a subject with hyperammonemia.

[0087] The term "hyperammonemia" refers to a condition in which plasma ammonia levels exceed normal, for example, greater than 30 μmol / L, or 50 μmol / L, or 80 μmol / L, or 100 μmol / L, or 120 μmol / L. In severe hyperammonemia, plasma ammonia levels exceed 1000 μmol / L.

[0088] Glucagon signaling pathway inhibitors Provided herein are glucagon signaling pathway antagonists, such as GCG inhibitors and GCGR antagonists, for the treatment of conditions or diseases characterized by hyperammonemia. In some embodiments, the antagonists are glucagon inhibitors, such as amylin and pramlintide. In some embodiments, the antagonists are GCGR inhibitors. In some embodiments, the GCGR antagonists are MK-0893, PF-06291874, LGD-6972, or LY2409021.

[0089] In some embodiments, the antagonist comprises an antibody capable of binding GCG or GCGR, or a fragment thereof. In some embodiments, the signal transduction pathway is inhibited by disrupting GCG or GCGR expression, for example, by using CRISPR technology or antisense, or by targeting downstream enzymes such as CaMKII.

[0090] In some embodiments, the GCG inhibitor or GCGR antagonist is an antisense molecule (GR-ASO), an antibody, a small molecule inhibitor, shRNA, siRNA, a peptide inhibitor (amylin, pramlintide), a DARPin, a spiegelmer, an aptamer, an engineered Fn3-type domain, or a derivative thereof.

[0091] Anti-GCGR antibodies, anti-GCG antibodies, and antibody fragments In some embodiments, the GCGR antagonist is an antibody or antibody fragment disclosed in U.S. Patent No. 8,545,847, the entire contents of which are incorporated herein by reference. The antibodies disclosed therein are provided in Table 1.

[0092] (Table 1) TIFF2023182691000001.tif97164

[0093] Additional GCGR antibodies or antibody fragments contemplated as useful herein include those disclosed in U.S. Patent Application Publication Nos. 5,770,445 and 7,947,809; European Patent Application No. EP2074149A2; European Patent No. EP0658200B1; U.S. Patent Publications 2009 / 0041784; 2009 / 0252727; and 2011 / 0223160; and PCT Publication WO2008 / 036341. Patents and publications are incorporated herein by reference in their entireties.

[0094] In some embodiments, the GCG inhibitor is an antibody or antibody fragment thereof, as disclosed in US 2016 / 0075778, the entirety of which is incorporated herein by reference. The antibodies disclosed therein are provided in Table 2.

[0095] (Table 2) TIFF2023182691000002.tif79164

[0096] Additional GCG antibodies or antibody fragments contemplated as useful herein include those disclosed in U.S. Patent Application Publication Nos. 4,206,199, 4,221,777, 4,423,034, 4,272,433, 4,407,965, 5,712,105, and PCT Publication Nos. WO2007 / 124463 and WO2013 / 081993.

[0097] Antibody fragments include any fragment with the required target specificity, such as, for example, antibody fragments produced by modification of whole antibodies (e.g., enzymatic digestion) or antibody fragments synthesized de novo using recombinant DNA methodologies (scFv, single domain antibodies, DVDs (dual variable domain immunoglobulins), or dAbs (single variable domain antibodies)), or antibody fragments identified using human phage or yeast display libraries (see, e.g., McCafferty et al. (1990) Nature 348:552-554). Alternatively, antibodies can be isolated from mice producing human, human-mouse, human-rat, and human-rabbit chimeric antibodies using standard immunization and antibody isolation methods, including, but not limited to, generating hybridomas or using B-cell screening techniques such as SLAM. Immunoglobulin binding domains include, but are not limited to, immunoglobulin heavy (V) domains. H ) chain or light (V L Alternatively, by immunizing humans, isolating antigen-positive B cells, cloning the cDNAs encoding the heavy and light chains, and co-expressing them in cells such as CHO.

[0098] The term "antibody" as used herein refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragment thereof that specifically binds and recognizes an antigen. Recognized immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant regions, as well as various immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes: IgG, IgM, IgA, IgD, and IgE, respectively. Within each IgG class, there are different isotypes (e.g., IgG1, IgG2, IgG3, IgG4). Typically, the antigen-binding region of an antibody is most critical in determining binding specificity and affinity.

[0099] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, each pair having one light chain (approximately 25 kD) and one heavy chain (approximately 50-70 kD). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids that is primarily responsible for antigen recognition. The "variable light chain" (VLC) is a L ), and variable heavy chain (V H ) refer to these light and heavy chains respectively.

[0100] Antibodies exist as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. For example, pepsin digests antibodies below the disulfide bonds in the hinge region, itself splitting them into V fragments by disulfide bonds. H -C H The resulting dimer of Fab, a light chain linked to Fab', is generated, F(ab)'2. F(ab)'2 can be reduced under mild conditions to cleave the disulfide bond in the hinge region, thereby converting the F(ab)'2 dimer into a Fab' monomer. The Fab' monomer is essentially Fab with part of the hinge region. While various antibody fragments have been defined with respect to the digestion of intact antibodies, those skilled in the art will appreciate that such fragments can be synthesized de novo, either chemically or using recombinant DNA methodologies.

[0101] Methods for preparing antibodies useful in accordance with the methods herein are known in the art. See, for example, Kohler & Milstein (1975) Nature 256:495-497; Harlow & Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Lab., Cold Spring Harbor, NY). Genes encoding the heavy and light chains of an antibody of interest can be cloned from cells; for example, genes encoding monoclonal antibodies can be cloned from hybridomas and used to generate recombinant monoclonal antibodies. Monoclonal antibodies can be humanized using standard cloning of CDR regions into human frameworks. Gene libraries encoding the human heavy and light chains of monoclonal antibodies can also be generated from hybridomas or plasma cells. Random combination of heavy and light chain gene products generates a large pool of antibodies with different antigen specificities. Techniques for the production of single chain antibodies or recombinant antibodies (U.S. Pat. Nos. 4,946,778; 4,816,567) can be adapted to produce antibodies for use in the methods disclosed herein. Also, transgenic mice, or other organisms such as other mammals, may be used to express human, human-mouse chimeric, human-rat chimeric, human-rabbit chimeric, or humanized antibodies. Alternatively, phage display or yeast display technologies can be used to identify human antibodies and heteromeric Fab fragments that specifically bind to a selected antigen.

[0102] Immunoconjugates The present disclosure encompasses the treatment of hyperammonemia with a human anti-GCGR monoclonal antibody (or human anti-GCG monoclonal antibody) conjugated to a therapeutic moiety, such as an agent capable of lowering blood ammonia levels or addressing another symptom of hyperammonemia (an "immunoconjugate"). The type of therapeutic moiety that can be conjugated to an anti-GCGR antibody takes into account the condition being treated and the desired therapeutic effect to be achieved. For example, an agent such as a glucagon receptor pathway antagonist or a second GCGR or GCG inhibitor may be conjugated to a GCGR antibody to lower blood ammonia levels and / or maintain normal blood ammonia levels. Alternatively, if the desired therapeutic effect is to reduce any other symptoms or conditions associated with glutamine or urea cycle disorders, it may be advantageous to conjugate an appropriate agent to the anti-GCGR antibody. Examples of suitable agents for forming immunoconjugates are known in the art.

[0103] multispecific antibodies Antibodies useful in accordance with the methods provided herein may be monospecific, bispecific, or heavy Specificity or multiplicity heavy It may be specific. heavy Specific antibodies may be specific for different epitopes of a single target polypeptide, or may contain antigen-binding domains specific for two or more target polypeptides. See, e.g., Tutt et al., (1991) J. Immunol. 147:60-69; Kufer et al., (2004) Trends Biotechnol. 22:238-244. Anti-GCGR antibodies can be linked to or co-expressed with another functional molecule, e.g., another peptide or protein. For example, an antibody or fragment thereof can be operably linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or other means) to one or more other molecular entities, e.g., another antibody or antibody fragment, to produce a second antibody or antibody fragment with a second binding specificity. heavy Specificity or multiplicity heavyFor example, if one arm of an immunoglobulin is specific for human GCGR or a fragment thereof, two heavy Specific antibodies are contemplated, with the other arm of the immunoglobulin being specific for a second therapeutic target or conjugated to a therapeutic moiety. In certain embodiments, one arm of the immunoglobulin is specific for an epitope in the N-terminal domain of hGCGR or a fragment thereof, and the other arm of the immunoglobulin is specific for an epitope in one of the EC loops of hGCGR or a fragment thereof. In certain embodiments, one arm of the immunoglobulin is specific for one EC loop or a fragment thereof, and the second arm is specific for a second EC loop or a fragment thereof. In certain embodiments, one arm of the immunoglobulin is specific for one epitope in one EC loop of hGCGR, and the other arm is specific for a second epitope in the same EC loop of hGCGR.

[0104] Exemplary diastereolytic enzymes that can be used in accordance with the methods described herein heavy The specific antibody format is the first immunoglobulin (Ig) C H 3 domain and second IgC H the use of three domains, wherein the first and second IgC H The three domains differ from each other by at least one amino acid, and wherein the difference in at least one amino acid is greater than or equal to two domains lacking an amino acid difference. heavy Compared to specific antibodies, heavy In one embodiment, the first Ig C H The 3 domain binds to protein A and the second Ig C H The C3 domain contains mutations that reduce or abolish Protein A binding, such as the H95R modification (according to IMGT exon numbering, H435R in EU numbering). H 3 may further contain a Y96F modification (Y436F in EU according to IMGT). HAdditional modifications that may be found in 3 include: D16E, L18M, N44S, K52N, V57M, and V82I for IgG1 antibodies (D356E, L358M, N384S, K392N, V397M, and V422I in EU by IMGT), N44S, K52N, and V821 for IgG2 antibodies (N384S, K392N, and V422I in EU by IMGT), and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I for IgG4 antibodies (Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I in EU by IMGT). heavy Variations in specific antibody formats are contemplated within the scope of this disclosure.

[0105] Antibody screening and selection Screening and selection of preferred antibodies useful according to the methods provided herein can be performed by various methods known in the art. Initial screening for the presence of monoclonal antibodies specific to the target antigen may be performed, for example, through the use of ELISA-based methods. A secondary screen is preferably performed to identify and select desirable monoclonal antibodies for use in constructing antibody-drug conjugates. The secondary screen may be performed by any suitable method known in the art. One preferred method, called "Biosensor Modification-Assisted Profiling" ("BiaMAP"), is described in U.S. Publication No. 2004 / 0101920, which is specifically incorporated by reference in its entirety. BiaMAP allows for the rapid identification of hybridoma clones producing monoclonal antibodies with desirable properties. More specifically, monoclonal antibodies are classified into distinct epitope-related groups based on evaluation of antibody:antigen interactions. Antibodies capable of blocking either the ligand or the receptor can be identified by cell-based assays, such as luciferase assays utilizing a luciferase gene under the control of an NFκB-driven promoter or a cAMP-responsive promoter. Stimulation of GCGR by glucagon leads to a signal through NFκB / cAMP / CREB, thus increasing luciferase levels in the cell. Blocking antibodies are identified as antibodies that block glucagon induction of luciferase activity.

[0106] treatment group The therapeutic methods provided herein are useful for treating individuals with urea cycle disorders or hyperammonemia-related conditions or diseases. In some embodiments, the subject suffers from congenital hyperammonemia, such as a deficiency in a urea cycle enzyme selected from the group consisting of carbamyl phosphate synthetase (CPS1), N-acetylglutamate synthetase (NAGS), ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL) and arginase (AR1), or a deficiency in a urea cycle transporter selected from ornithine translocase (ORNT1) and citrin. Other congenital disorders that can cause hyperammonemia include propionic acidemia, isolated methylmalonic acidemia, isovaleric acidemia, carbonic anhydrase Va deficiency, lysinuric protein intolerance, carnitine palmitoyltransferase II deficiency, carnitine-acylcarnitine translocase, pyrroline-5-carboxylate synthetase deficiency, pyruvate carboxylase deficiency, ornithine aminotransferase deficiency, carbonic anhydrase Va deficiency, hyperinsulinemia-hyperammonemia syndrome, mitochondrial disorders, glutamine synthetase deficiency, fatty acid oxidation disorders (e.g., short-chain acyl-CoA dehydrogenase deficiency, medium-chain acyl-Coenzyme A dehydrogenase deficiency, multiple acyl-Coenzyme A dehydrogenase deficiency, very-long-chain acyl-Coenzyme A dehydrogenase deficiency), and hyperinsulinemia-hyperammonemia syndrome (familial hyperinsulinism).

[0107] In some embodiments, hyperammonemia is acquired, for example, caused by liver disease and its complications, treatment with therapeutic agents (L-asparaginase or pegaspargase), 5-pentanoic acid, valproic acid, corticosteroids, or cyclophosphamide, or herpes simplex or hepatitis B infection. Additional causes of hyperammonemia, i.e., acquired hyperammonemia, include, for example, liver disease and its complications, such as hepatic encephalopathy in subjects with advanced liver disease, hepatic halitosis (a late sign of liver failure), hepatic vascular bypass, and biliary atresia, administration of toxic levels of valproic acid, corticosteroids, or cyclophosphamide, herpes simplex infection, and gastrointestinal bacterial overgrowth. Hyperammonemia can also be caused by infection with urease-producing organisms.

[0108] Additionally, hyperammonemia can be caused by total parenteral nutrition (with relative arginine deficiency), nutritional carnitine deficiency, cystoscopy with glycine-containing solutions, after lung / bone marrow transplantation, congenital anomalies of the blood vessels, or transient hyperammonemia in newborns.

[0109] In some embodiments, elevated ammonia levels are detected in the serum of the subject. In some embodiments, excess glutamine is detected in the serum of the subject.

[0110] Therapeutic Administration and Formulations For example, therapeutic compositions containing glucagon / GCGR antagonists, such as anti-GCGR antibodies, are useful in accordance with the methods provided herein. Administration of therapeutic compositions according to the methods described herein will be via a suitable route, including, but not limited to, intravenous, subcutaneous, intramuscular, intrathecal, intracerebral, intracerebroventricular, intranasal, or oral, with suitable transporters, excipients, and other agents incorporated into the formulation to provide improved transport, delivery, tolerance, etc. Numerous suitable formulations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, a formulary known in all pharmaceutical chemistry circles. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsifiable concentrates (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0111] The dose of the antibody may vary depending on the age and size of the subject to be administered, the target disease, condition, route of administration, etc. When the antibody is used to treat hyperammonemia or to reduce blood ammonia levels associated with a urea cycle disorder in a subject, it is typically advantageous to administer the antibody intravenously at a dose of about 0.01 to about 30 mg per kg of body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg per kg of body weight. The frequency and duration of treatment can be adjusted depending on the severity of the condition and response to treatment. In certain embodiments, the antibody or antigen-binding fragment thereof may be administered as an initial dose of at least about 0.1 mg to about 800 mg, about 1 to about 500 mg, about 5 to about 300 mg, or about 10 to about 200 mg, about 100 mg, or about 50 mg.

[0112] In certain embodiments, the initial dose may be followed by administration of a second or multiple subsequent doses of the antibody or antigen-binding fragment thereof, in an amount that may be about the same as the initial dose or may be a smaller dose than the initial dose, wherein the subsequent doses may be separated by at least 1 to 3 days; at least 1 week, at least 2 weeks, at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks; or until hyperammonemia has resolved.

[0113] Various delivery systems are known and can be used to administer pharmaceutical compositions containing antibodies, such as liposomes, microparticles, microcapsules, encapsulation in recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis, etc. (See, e.g., Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, depot formulations, aerosols, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, intrathecal, intraventricular, and oral routes. The compositions can be administered by any convenient route, such as by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral, rectal, and intestinal mucosa), and can be administered together with other biologically active agents. Administration can be systemic or local.

[0114] Pharmaceutical compositions can also be delivered in vesicles, in particular liposomes (see, eg, Langer (1990) Science 249:1527-1533).

[0115] In certain circumstances, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used. In another embodiment, a polymeric material can be used. In yet another embodiment, the sustained release system can be placed in proximity to the target of the composition, thus requiring only a fraction of the systemic dose.

[0116] Injectable preparations may include administration forms such as intravenous, subcutaneous, intradermal, and intramuscular injections, infusions, and the like. These injectable preparations may be prepared by known methods. For example, injectable preparations may be prepared by dissolving, suspending, or emulsifying the above-described antibody or its salt in a sterile aqueous or oily medium conventionally used for injections. Aqueous media for injection include, for example, saline, isotonic solutions containing glucose, and other auxiliary agents, which may be used in combination with suitable solubilizers such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Oily media include, for example, sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injection solution prepared in this manner is preferably filled into an appropriate ampule.

[0117] The pharmaceutical compositions useful in the present disclosure can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, pen delivery devices readily find use in delivering the pharmaceutical compositions useful in the methods described herein. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Rather, disposable pen delivery devices are sold pre-filled with the pharmaceutical composition held in a reservoir inside the device. Once the reservoir is emptied of pharmaceutical composition, the entire device is discarded.

[0118] Numerous reusable pen and autoinjector delivery devices find use in the subcutaneous delivery of pharmaceutical compositions useful according to the methods described herein. Examples include the AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), the DISETRONIC™ pen (Disetronic Medical Systems, Burgdorf, Switzerland), the HUMALOG MIX 75 / 25™ pen, the HUMALOG™ pen, the HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), the NOVOPEN™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), the NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), the BD™ pen (Becton Dickinson, Franklin Lakes, NJ), the OPTIPEN™, the OPTIPEN PRO™, the OPTIPEN IV ... Examples of disposable pen-type delivery devices that find use in the subcutaneous delivery of pharmaceutical compositions useful according to the methods described herein include, but are by no means limited to, the SOLOSTAR Pen (sanofi-aventis), FLEXPEN (Novo Nordisk), and KWIKPEN (Eli Lilly), SURECLICK Autoinjector (Amgen, Thousand Oaks, California), PENLET (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP) and HUMIRA Pen (Abbott Labs, Abbott Park, Ill.), to name just a few.

[0119] Advantageously, the above-mentioned pharmaceutical compositions for oral or parenteral use are prepared in dosage forms with unit doses suitable for adjusting the dose of the active ingredient. Such dosage forms in unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the above-mentioned antibody contained per dosage form in a unit dose is generally about 5 to about 750 mg. In particular, in the form of injection, the above-mentioned antibody is preferably contained in an amount of about 5 to about 100 mg, and for other dosage forms, the antibody is preferably contained in an amount of about 10 to about 250 mg.

[0120] Combination therapy In many embodiments, the GCG inhibitors or GCGR antagonists useful herein may be administered in combination with one or more additional compounds, therapeutic agents, or therapies. The combination therapy may be simultaneous (or concomitant) or sequential. In some aspects, the additional compound (or therapeutic agent) is formulated in the same pharmaceutical composition as the GCG inhibitor or GCGR antagonist. In some aspects, the additional compound is administered before, after, alone, or in combination with the GCG inhibitor or GCGR antagonist.

[0121] In some embodiments, the glucagon signaling pathway antagonist is administered with at least one additional therapeutic agent selected from the following: insulin, a non-absorbable antibiotic (rifaximin or lactulose), sodium phenylbutyrate, sodium benzoate, sodium phenylacetate, glycerol phenylbutyrate, carbamyl glutamate (Carbaglu®), a second GCG inhibitor, and a second GCGR antagonist. In some embodiments, the glucagon signaling pathway antagonist is administered with hemodialysis or continuous renal replacement. In some embodiments, the glucagon signaling pathway antagonist is administered with L-citrulline or L-arginine free base. In some embodiments, the glucagon signaling pathway antagonist is administered with antioxidants or electrolytes. In some embodiments, the glucagon signaling pathway antagonist is administered with an amino acid formulation selected from Cyclinex, EAAs, UCD I & II, and individual branched-chain amino acids.

[0122] The additional therapeutically active ingredient(s) may be administered before, simultaneously with, or after administration of a glucagon signaling pathway antagonist, such as a GCG inhibitor or GCGR antagonist. For purposes of this disclosure, such administration regimens are considered administration of a glucagon signaling pathway antagonist "in combination" with the second therapeutically active ingredient.

[0123] Dosing regimen According to certain embodiments described herein, multiple doses of a glucagon / GCGR signaling pathway antagonist may be administered to a subject over a defined time course. The method includes sequentially administering multiple doses of a glucagon / GCGR signaling pathway antagonist to a subject. As used herein, "sequentially administering" means that each dose of the antagonist is administered to the subject at a different time point, for example, on a different day separated by a predetermined interval (e.g., hour, day, week, or month). The method described herein includes administering to the subject a single initial dose of a glucagon / GCGR signaling pathway antagonist, followed by one or more secondary doses of the glucagon / GCGR signaling pathway antagonist, and optionally, followed by one or more tertiary doses of the glucagon / GCGR signaling pathway antagonist.

[0124] The terms "initial dose," "secondary dose," and "tertiary dose" refer to the time sequence of administration of a glucagon / GCGR signaling pathway antagonist useful herein. Thus, an "initial dose" is a dose administered at the beginning of a treatment regimen (also referred to as a "baseline dose"), a "secondary dose" is a dose administered after the initial dose, and a "tertiary dose" is a dose administered after the secondary dose. The initial dose, secondary dose, and tertiary dose all contain the same amount of glucagon / GCGR signaling pathway antagonist, but typically may differ from one another in terms of administration frequency. However, in certain embodiments, the amount of glucagon / GCGR signaling pathway antagonist contained in the initial dose, secondary dose, and / or tertiary dose differs from one another (e.g., adjusted accordingly) during the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered as "loading doses" at the beginning of a treatment regimen, with subsequent doses administered on a less frequent basis (e.g., "maintenance doses").

[0125] Pharmaceutical Composition The methods disclosed herein contemplate the use of pharmaceutical compositions containing at least a therapeutically effective amount of an active agent useful for treating hyperammonemia or urea cycle disorders, such as a glucagon signaling pathway antagonist, and a pharmaceutically acceptable transporter. The term "pharmaceutically acceptable" means approved by federal or state regulatory agencies or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, more specifically in humans. The term "transporter" refers to a diluent, adjuvant, excipient, or vehicle administered with a therapeutic agent. Such pharmaceutical transporters can be sterile liquids, such as water and oils (including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.). Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. If desired, the compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The compositions can also be formulated as suppositories with traditional binders and carriers such as triglycerides. Oral formulations can contain standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.

[0126] In one embodiment, the composition is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to humans.If necessary, the composition can also contain a solubilizing agent such as lidocaine and a local anesthetic to reduce pain at the injection site.When the composition is administered by injection, it can be dispensed in an infusion bottle containing sterile pharmaceutical-grade water or saline.When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed before administration.

[0127] Active agents useful according to the methods described herein can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed with free carboxyl groups, such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0128] The amount of active agent that will be effective in treating hyperammonemia can be determined by standard clinical techniques based on the present disclosure. Additionally, in vitro assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in a formulation will also depend on the route of administration and the severity of the condition, and should be decided according to the judgment of the practitioner and each subject's circumstances. However, suitable dosage ranges for intravenous administration are generally about 20 micrograms to 2 grams of active compound per kilogram of body weight. Suitable dosage ranges for intranasal administration are generally about 0.01 pg to 1 mg per kilogram of body weight. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0129] For systemic administration, a therapeutically effective dose can be estimated initially from in vitro assays. For example, a dose may be estimated based on the IC 50The initial dose can be estimated from in vivo data, for example, animal models, using techniques well known in the art. Those skilled in the art can easily optimize human administration based on animal data.

[0130] Dosage amount and interval can be individually adjusted to provide sufficient plasma level of compound to maintain therapeutic effect.In the case of local administration or selective uptake, effective local concentration of compound may not be related to plasma concentration.Those skilled in the art can optimize therapeutically effective local dose without undue experimentation.

[0131] The amount of compound administered will, of course, depend on the subject being treated, and on the subject's weight, the severity of the affliction, the manner of administration, and the judgment of the prescribing physician. Therapy may be repeated intermittently, whether or not symptoms are detectable. Therapy may be provided alone or in combination with other agents.

[0132] kit Also provided herein are articles of manufacture that include packaging material and a pharmaceutical product contained within the packaging material, wherein the pharmaceutical product comprises at least one glucagon signaling pathway antagonist useful according to the methods disclosed herein, and wherein the packaging material comprises a label or package insert that indicates the glucagon signaling pathway antagonist can be used to treat a urea cycle disorder or a condition or disease characterized by hyperammonemia.

[0133] While the present invention has been particularly shown and described with reference to numerous embodiments, those skilled in the art will understand that changes may be made in form and detail to the various embodiments disclosed herein without departing from the spirit and scope of the invention, and that the various embodiments disclosed herein are not intended to serve as limitations on the scope of the claims. [Example]

[0134] The following examples are provided so that those skilled in the art will have a complete disclosure and description of how to practice the methods disclosed herein. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are by weight, molecular weight is average molecular weight, and temperature is in °C (Celsius) at or near atmospheric pressure.

[0135] Example 1: Evaluation of GCGR antagonists in a mouse model of urea cycle disorder (H4H1327P) The effects of mAb1, a GCGR monoclonal antibody having the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 86 / 88 (H4H1327P), on body weight, survival rate, plasma ammonia level, and blood glucose level were investigated in a mouse model of urea cycle disorder, ornithine transcarbamylase mutant (OTC) spf-ash ) determined in mice. spf-ash The mutation is a missense transition from G to A at the last nucleotide of exon 4 in the mouse Otc gene. The mutation changes arginine to histidine (R129H), resulting in inefficient mRNA splicing at this site and a site located 48 bases within the adjacent intron. The allele is hypomorphic, resulting in the retention of 5–10% of wild-type hepatic ornithine transcarbamylase enzyme activity. Otc mutant mice are viable, fertile, and show no overt disease, but are smaller than wild-type mice. A high-protein diet (40% vs. 21% in a normal diet) in Otc mutant mice has been shown to cause hyperammonemia and 30% mortality within one week (Yang et al., A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice. Nat Biotechnol. 2016 34:334-8). In this experiment, Otc mutant mice were fed a 30% protein diet, which induced mild hyperammonemia and 60% mortality within 9 weeks.

[0136] Forty-one wild-type and 34 Otc mutant mice were used in this study. To establish a baseline for blood glucose measurements, non-fasting blood glucose was verified in blood drawn from the tail of each mouse at 9:00 AM on day 0 using an ACCU-CHEK® Compact Plus (Roche). Wild-type and Otc mutant mice were each divided into two treatment groups based on their blood glucose levels, ensuring that mean glucose levels were approximately equal between treatment groups. From day 0 to the end of the study, half of the wild-type (n = 21) and Otc mutant (n = 17) mice received weekly subcutaneous injections of mAb1 at 10 mg / kg, while the other half of the wild-type (n = 20) and Otc mutant (n = 17) mice received weekly subcutaneous injections of hIgG4 isotype control at 10 mg / kg. All mice were placed on a high-protein diet (30% protein by weight) starting on day 10 for the duration of the study. Non-fasting blood glucose and body weight were measured weekly. The mean ± SEM of blood glucose levels at each time point was calculated for each group and is shown in Table 3 and Figure 1. The mean ± SEM of body weight change from baseline (day 0) at each time point was calculated for each group, as shown in Table 4 and Figure 2. Plasma was collected at baseline and at weeks 2, 3, 4, 6, 7, and 8, and ammonia levels were determined. The mean ± SEM of plasma ammonia levels at each time point was calculated for each group and is shown in Table 5 and Figure 3. Animal deaths were recorded daily. Survival curves for each group are shown in Figure 4. Statistical analysis of blood glucose, body weight, and plasma ammonia was performed by two-way ANOVA comparing each treatment group within each genotype, followed by Bonferroni post-tests.

[0137] Results and conclusions: mAb1-treated wild-type and Otc mutant mice showed decreased blood glucose after mAb1 administration compared with isotype control-treated animals (between weeks 1 and 9), confirming the glucose-lowering efficacy of mAb1 (Table 3 and Figure 1). The high-protein diet induced approximately 15% weight loss in isotype control-treated Otc mutant mice, whereas mAb1-treated Otc mutant mice were protected from diet-induced weight loss (Table 4 and Figure 2). The high-protein diet-induced increase in plasma ammonia levels was smaller in mAb1-treated Otc mutant mice compared with isotype control-treated Otc mutant mice (Table 5 and Figure 3). None of the wild-type mice died during the study, regardless of treatment. At the end of the study, 88% (15 of 17) of the mAb1-treated Otc mutant mice were alive, compared with 47% (8 of 17) of the isotype control-treated Otc mutant mice (Figure 4).

[0138] These data suggest that in a mouse model of urea cycle disorders, mAb1 protects against hyperammonemia, excessive weight loss, and death in Otc mutant mice on a high-protein diet.

[0139] (Table 3) Blood glucose levels TIFF2023182691000003.tif105149

[0140] (Table 4) Weight change from baseline TIFF2023182691000004.tif105149

[0141] Table 5. Plasma ammonia levels TIFF2023182691000005.tif83149

[0142] A second study of different design was conducted to determine the effect of H4H1327P on mice with urea cycle disorders, as follows.

[0143] The effects of H4H1327P on plasma ammonia levels, survival rate, body weight, and blood glucose levels were compared with those of the ornithine transcarbamylase mutant (OTC), a mouse model of urea cycle disorders. spf-ash ) mice. spf-ash ) mice were initially fed a high-protein diet (30% protein vs. 21% protein in a normal diet). Two days after starting the diet, mild hyperammonemia was observed in Otc mutant mice before antibody treatment began.

[0144] Twenty wild-type and twenty Otc mutant mice were used in this study. To establish a baseline for plasma ammonia measurements, non-fasting ammonia levels were tested in plasma collected from the submandibular gland of each mouse at 9:00 AM on Day 0 using an ADVIA® 1800 blood chemistry analyzer (Bayer, Leverkusen, Germany). All mice received a high-protein diet (30% protein by weight) from Day 0 throughout the study. Wild-type and Otc mutant mice were divided into two treatment groups based on their plasma ammonia levels on Day 2, ensuring that the mean ammonia levels of each treatment group for each genotype were approximately equal. From Day 2 until the end of the study, half of the wild-type (n = 10) and Otc mutant (n = 10) mice received weekly subcutaneous injections of H4H1327P at 10 mg / kg. The other half of the wild-type (n = 10) and Otc mutant (n = 10) mice received weekly subcutaneous injections of 10 mg / kg hIgG4 isotype control. Plasma was collected at baseline, day 2, and every 5–12 days thereafter to determine ammonia levels. The mean ± SEM of plasma ammonia levels at each time point was calculated for each group and is shown in Table 6 and Figure 5. Animal deaths were recorded daily. The survival curves for each group are shown in Figure 6. Body weight and nonfasting blood glucose were measured weekly. The mean ± SEM of body weight change from baseline (day 0) at each time point was calculated and is shown in Table 7 and Figure 8. The mean ± SEM of blood glucose levels at each time point was calculated for each group and is shown in Table 8 and Figure 8. Statistical analysis of blood glucose, body weight, and plasma ammonia was performed by two-way ANOVA comparing each treatment group within each genotype, followed by Bonferroni post-tests.

[0145] The high-protein diet increased plasma ammonia levels in Otc mutant mice from 65.2 to 129.4 μmol / L within 2 days. Otc mutant mice treated with the isotype control showed even higher mean plasma ammonia levels of 271–359 μmol / L over the study period, whereas mean plasma ammonia levels in H4H1327P-treated Otc mutant mice remained within the range of 105–232 μmol / L (Table 6 and Figure 5). At the end of the study, 80% (8 of 10) of the H4H1327P-treated Otc mutant mice were alive, compared with 40% (4 of 10) of the isotype control-treated Otc mutant mice (Figure 6). None of the wild-type mice died during the study, regardless of treatment. The high-protein diet induced an 18.5% weight loss in isotype control-treated Otc mutant mice, while H4H1327P-treated Otc mutant mice showed a 20.9% increase in body weight at the end of the study (Table 7 and Figure 7). H4H1327P-treated wild-type and Otc mutant mice showed a decrease in blood glucose after H4H1327P administration compared to isotype control-treated animals, confirming the glucose-lowering efficacy of H4H1327P (Table 8 and Figure 8).

[0146] Collectively, H4H1327P was able to ameliorate the hyperammonemia, excessive weight loss, and death induced by a high-protein diet in Otc mutant mice. These data indicate that H4H1327P may be a useful option for patients with urea cycle disorders.

[0147] Table 6. Plasma ammonia levels TIFF2023182691000006.tif135149

[0148] (Table 7) Weight change from baseline TIFF2023182691000007.tif135149

[0149] (Table 8) Blood glucose levels TIFF2023182691000008.tif135149

[0150] Array information SEQUENCE LISTING <110> Regeneron Pharmaceuticals, Inc. <120> METHODS OF TREATING UREA CYCLE DISORDERS BY INTERFERING WITH GLUCAGON RECEPTOR SIGNALING <150> US 62 / 548,632 <151> 2017-08-22 <160> 308 <170> PatentIn version 3.5 <210> 1 <211> 375 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 1 caggtccagt tggtacagtc tggggctgac gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg tttccggaca tatcctcact gatttatcca tgcactgggt gcgacagcct 120 cctggaaaag gacttgagtg gatggcaggt tttgatcctg aagaaggtaa aataatctac 180 gcacagaagt tccagggcag agtcaccatg accgaggaca catctacaga cacagcctac 240 atggagctga gcagcctgag atctggggac acggccgttt attactgtgc aacaagcgat 300 attttgactg ggtattatag agactactac ggtttggacg tctggggcca agggaccacg 360 ctcaccgtct cctca 375 <210> 2 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 2 Gln Val Gln Leu Val Gln Ser Gly Ala Asp Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Val Ser Gly His Ile Leu Thr Asp Leu 20 25 30 Ser Met His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Ala Gly Phe Asp Pro Glu Glu Gly Lys Ile Ile Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Gly Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Ser Asp Ile Leu Thr Gly Tyr Tyr Arg Asp Tyr Tyr Gly Leu 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 115 120 125 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 3 ggacatatcc tcactgattt atcc 24 <210> 4 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 4 Gly His Ile Leu Thr Asp Leu Ser 1 5 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 5 tttgatcctg aagaaggtaa aata 24 <210> 6 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 6 Phe Asp Pro Glu Glu Gly Lys Ile 1 5 <210> 7 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 7 gcaacaagcg atattttgac tgggtattat agagactact acggtttgga cgtc 54 <210> 8 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 8 Ala Thr Ser Asp Ile Leu Thr Gly Tyr Tyr Arg Asp Tyr Tyr Gly Leu 1 5 10 15 Asp Val <210> 9 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 9 gatattgtga tgactcagtc tccactcttc ctgcccgtca cccctggaga gccggcctc 60 atctcctgca ggtctagtca gagcctcctg catagtaaag gatacaacta tttggattgg 120 tacctgcaga agccagggca gtctccacaa ctcctgatct atttgggttc taatcgggcc 180 tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240 agcagagtgg aggctgaaga tgttggggtt tattactgca tgcaaactct acaaactcct 300 cggacgttcg gccaagggac caaggtggaa atcaaa 336 <210> 10 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 10 Asp Ile Val Met Thr Gln Ser Pro Leu Phe Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Lys Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Thr 85 90 95 Leu Gln Thr Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 11 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 11 cagagcctcc tgcatagtaa aggatacaac tat 33 <210> 12 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 12 Gln Ser Leu Leu His Ser Lys Gly Tyr Asn Tyr 1 5 10 <210> 13 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 13 ttgggttct 9 <210> 14 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 14 Leu Gly Ser 1 <210> 15 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 15 atgcaaactc tacaaactcc tcggacg 27 <210> 16 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 16 Met Gln Thr Leu Gln Thr Pro Arg Thr 1 5 <210> 17 <211> 375 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 17 caggtccagt tggtacagtc tggggctgac gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg tttccggaca tatcctcact gatttatcca tgcactgggt gcgacaggct 120 cctggaaaag ggcttgagtg gatgggaggt tttgatcctg aagaaggtga aataatctac 180 gcacagaagt tccagggcag agtcaccatg accgaggaca catctacaga cacagcctac 240 atggagctga gcagcctgag atctggggac acggccgttt attactgtgc aacaagcgat 300 attttgactg gttattatag agactactac ggtttggacg tctggggcca agggaccacg 360 ctcaccgtct cctca 375 <210> 18 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 18 Gln Val Gln Leu Val Gln Ser Gly Ala Asp Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Val Ser Gly His Ile Leu Thr Asp Leu 20 25 30 Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Gly Phe Asp Pro Glu Glu Gly Glu Ile Ile Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Gly Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Ser Asp Ile Leu Thr Gly Tyr Tyr Arg Asp Tyr Tyr Gly Leu 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 115 120 125 <210> 19 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 19 ggacatatcc tcactgattt atcc 24 <210> 20 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 20 Gly His Ile Leu Thr Asp Leu Ser 1 5 <210> 21 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 21 tttgatcctg aagaaggtga aata 24 <210> 22 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 22 Phe Asp Pro Glu Glu Gly Glu Ile 1 5 <210> 23 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 23 gcaacaagcg atattttgac tggttattat agagactact acggtttgga cgtc 54 <210> 24 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 24 Ala Thr Ser Asp Ile Leu Thr Gly Tyr Tyr Arg Asp Tyr Tyr Gly Leu 1 5 10 15 Asp Val <210> 25 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 25 gatattgtga tgactcagtc tccactcttc ctgcccgtca cccctggaga gccggcctcc 60 atctcctgca gtctagtca gagcctcctg catagtaaag gatacaacta tttggattgg 120 tacctgcaga agccaggggca gtctccacaa ctcctgatct atttggttc taatcggggcc 180 tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagatttac actgaaaatc 240 agcagagtgg aggctgaga tgttggggtt tattactgca tgcaactct acaactcct 300 cggacgttcg gccaagggac caggtggaa atcaa 336 <210> 26 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 26 Asp Ile Val Met Thr Gln Ser Pro Leu Phe Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Gln Ser Leu Leu His Ser 20 25 30 Lys Gly Tyr Asn Tyrole Leu Asp Trp Tyr Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Thr 85 90 95 Leu Gln Thr Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 27 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 27 cagagcctcc tgcatagtaa aggatacaac tat 33 <210> 28 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 28 Gln Ser Leu Leu His Ser Lys Gly Tyr Asn Tyr 1 5 10 <210> 29 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 29 ttgggttct 9 <210> 30 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 30 Leu Gly Ser 1 <210> 31 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 31 atgcaaactc tacaaactcc tcggacg 27 <210> 32 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 32 Met Gln Thr Leu Gln Thr Pro Arg Thr 1 5 <210> 33 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 33 gaggagcaac tggtggagtc tgggggagac ttggtacagc ctggagggtc cctaagactc 60 tcctgtgcag cctctggatt cactcagt agttatgaaa tgactgggt ccgccaggct 120 ccagggaagg ggctggagtg gtttcatac attagtagag gtggtagtct gatacactac 180 acagactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ttcactgtat 240 ctgcaaatga acagcctgag agccgaggac acggctgttt attactgtgt gagagaccca 300 gcagctcgtt atcattatta ttcacggt atggacgtct ggggccaagg gaccacggtc 360 accgtctcct ca 372 <210> 34 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 34 Glu Glu Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Tyr 20 25 30 Glu Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Arg Gly Gly Ser Leu Ile His Tyr Thr Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Asp Pro Ala Ala Arg Tyr His Tyr Tyr Tyr His Gly Met Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 35 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 35 ggattcactc tcagtagtta tgaa 24 <210> 36 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 36 Gly Phe Thr Leu Ser Ser Tyr Glu 1 5 <210> 37 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 37 attagtagag gtggtagtct gata 24 <210> 38 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 38 Ile Ser Arg Gly Gly Ser Leu Ile 1 5 <210> 39 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 39 gtgagagacc cagcagctcg ttcattat tattatcacg gtatggacgt c 51 <210> 40 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 40 Val Arg Asp Pro Ala Ala Arg Tyr His Tyr Tyr Tyr Tyr His Gly Met Asp 1 5 10 15 Val <210> 41 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 41 gatattgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60 atctcctgca ggtctagtca gagcctcctg cacaataatg gatataacta tttggattgg 120 tatctgcaga agccagggca gtctccacag ctcctgatct atttggggttc tagtcggggcc 180 tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttat actgaaaatc 240 agcagagtgg aggctgaaga tgttggggtt tattactgca tgcaagctct acaaactccg 300 tggacgttcg gccgagggac caaggtggaa atcaaa 336 <210> 42 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 42 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Asn 20 25 30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Ser Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ile Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95 Leu Gln Thr Pro Trp Thr Phe Gly Arg Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 43 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 43 cagagcctcc tgcacaataa tggatataac tat 33 <210> 44 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 44 Gln Ser Leu Leu His Asn Asn Gly Tyr Asn Tyr 1 5 10 <210> 45 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 45 ttgggttct 9 <210> 46 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 46 Leu Gly Ser 1 <210> 47 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 47 atgcaagctc tacaaactcc gtggacg 27 <210> 48 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 48 Met Gln Ala Leu Gln Thr Pro Trp Thr 1 5 <210> 49 <211> 384 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 49 gaggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt agttatgaca tgcactgggt ccgccaggct 120 ccaggcaagg ggctggagtg ggtggcagtt atatcatctg atggacgtga taatactat 180 gtagactccg tgaagggccg attcaccatc tccagagaca actccaagaa cacgctttat 240 ctgcaaatga acagcctgag agctgaggac acggctgttt attactgtgc gaaagagatg 300 gtgtattacg atattttgac tggttatcat aactactacg gtatggacgt ctggggccaa 360 gggaccacgg tcaccgtctc ctca 384 <210> 50 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 50 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Asp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Ser Asp Gly Arg Asp Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Met Val Tyr Tyr Asp Ile Leu Thr Gly Tyr His Asn Tyr 100 105 110 Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 51 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 51 ggattcacct tcagtagtta tgac 24 <210> 52 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 52 Gly Phe Thr Phe Ser Ser Tyr Asp 1 5 <210> 53 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 53 atatcatctg atggacgtga taaa 24 <210> 54 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 54 Ile Ser Ser Asp Gly Arg Asp Lys 1 5 <210> 55 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 55 gcgaaagaga tggtgtatta cgatattttg actggttatc ataactacta cggtatggac 60 gtc 63 <210> 56 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 56 Ala Lys Glu Met Val Tyr Tyr Asp Ile Leu Thr Gly Tyr His Asn Tyr 1 5 10 15 Tyr Gly Met Asp Val 20 <210> 57 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 57 gacatcgtga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgtc gggcgagtca gggcattaac aattatttag cctggtttca gcagaaacca 120 gggaaagccc ctaagtccct gatccatact gcatccagtt tgcaaagtgg ggtcccatca 180 aagttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240 gaagattttg caacttatta ctgccaacag tataatactt accctctcac tttcggcgga 300 gggaccaaag tggagatcaa acga 324 <210> 58 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 58 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Asn Asn Tyr 20 25 30 Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile 35 40 45 His Thr Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Lys Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Thr Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 59 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 59 cagggcatta acaattat 18 <210> 60 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 60 Gln Gly Ile Asn Asn Tyr 1 5 <210> 61 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 61 actgcatcc 9 <210> 62 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 62 Thr Ala Ser 1 <210> 63 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 63 caacagtata atacttaccc tctcact 27 <210> 64 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 64 Gln Gln Tyr Asn Thr Tyr Pro Leu Thr 1 5 <210> 65 <211> 381 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 65 caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt agttatgaca tgcactgggt ccgccaggct 120 ccaggcaagg ggctggagtg ggtggcagtt atatcatctg atggacgtga taaatactat 180 gtagactccg tgaagggccg attcaccatc tccagagaca actccaagaa cacgctttat 240 ctgcaaatga acagcctgag agctgaggac acggctgttt attactgtgc gaaagagatg 300 gtgtattacg atattttgac tggttatcat aactactacg gtatggacgt ctggggccaa 360 gggaccacgg tcaccgtctc c 381 <210> 66 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 66 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Asp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Ser Asp Gly Arg Asp Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Met Val Tyr Tyr Asp Ile Leu Thr Gly Tyr His Asn Tyr 100 105 110 Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115 120 125 <210> 67 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 67 gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgtc gggcgagtca gggcattaac aattatttag cctggtttca gcagaaacca 120 gggaaagccc ctaagtccct gatccatact gcatccagtt tgcaagtgg ggtcccatca 180 aagttcagcg gcagtggatc tgggacagat ttcactca ccatcagcag cctgcagcct 240 gaagattttg caacttatta ctgccacag tataatactt accctcac ttcggcgga 300 321 <210> 68 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 68 Asp With Gln Met Thr Gln Serving Pro Serving Leu Serving Ala Serving Val Gly 1 5 10 15 Asp Arg Val Thr Thr Cys Arg Only Be Gln Gly With Asn Tyr 20 25 30 Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile 35 40 45 His Thr Ala Ser Ser Leu Ser Gln Ser Gly Val Pro Ser Lys Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Thr Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 69 <211> 384 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 69 caggtgcagc tggtgcagtc tgggggaggc ttggtccagc ctggggggc cctgagactc 60 tcctgtgcag cctccggatt cacctttagt aactatttga tgaactgggt ccgccaggct 120 ccagggaagg ggctggagtg gctggccaac atacaggaag atggaattga gaaatactat 180 gtggactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat 240 ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagagagccc 300 tcccattacg atattttgac tggttatgac tactattacg gtatggacgt ctggggccaa 360 gggaccacgg tcaccgtctc ctca 384 <210> 70 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 70 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Leu Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Ala Asn Ile Gln Glu Asp Gly Ile Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Pro Ser His Tyr Asp Ile Leu Thr Gly Tyr Asp Tyr Tyr 100 105 110 Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 71 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 71 ggattcacct ttagtaacta tttg 24 <210> 72 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 72 Gly Phe Thr Phe Ser Asn Tyr Leu 1 5 <210> 73 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 73 atacaggaag atggaattga gaaa 24 <210> 74 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 74 Ile Gln Glu Asp Gly Ile Glu Lys 1 5 <210> 75 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 75 gcgagagagc cctcccatta cgatattttg actggttatg actactatta cggtatggac 60 gtc 63 <210> 76 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 76 Ala Arg Glu Pro Ser His Tyr Asp Ile Leu Thr Gly Tyr Asp Tyr Tyr 1 5 10 15 Tyr Gly Met Asp Val 20 <210> 77 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 77 gacatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 120 gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180 aggttcagcg gcagtggatc tgggacagaa ttcattctca cagtcagcag cctgcagcct 240 gaagactttg caacttatta ctgtctacag tataatagta acccattcac tttcggccct 300 gggaccaagg tggagatcaa acga 324 <210> 78 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 78 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Ile Leu Thr Val Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Asn Ser Asn Pro Phe 85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 79 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 79 cagggcatta gaaatgat 18 <210> 80 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 80 Gln Gly Ile Arg Asn Asp 1 5 <210> 81 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 81 gctgcatcc 9 <210> 82 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 82 Ala Ala Ser 1 <210> 83 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 83 ctacagtata atagtaaccc attcact 27 <210> 84 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 84 Leu Gln Tyr Asn Ser Asn Pro Phe Thr 1 5 <210> 85 <211> 381 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 85 gaggtgcagc tggtggagtc tgggggaggc ttggtccagc ctggggggc cctgagactc 60 tcctgtgcag cctccggatt cacctttagt aactatttga tgaactgggt ccgccaggct 120 ccagggaagg ggctggagtg gctggccaac atacaggaag atggaattga gaaatactat 180 gtggactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat 240 ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagagagccc 300 tcccattacg atattttgac tggttatgac tactattacg gtatggacgt ctggggccaa 360 gggaccacgg tcaccgtctc c 381 <210> 86 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 86 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Leu Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Ala Asn Ile Gln Glu Asp Gly Ile Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Pro Ser His Tyr Asp Ile Leu Thr Gly Tyr Asp Tyr Tyr 100 105 110 Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115 120 125 <210> 87 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 87 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 120 gggaaagccc ctaagcgcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180 aggttcagcg gcagtggatc tgggacagaa ttcattctca cagtcagcag cctgcagcct 240 gaagactttg caacttatta ctgtctacag tataatagta acccattcac ttcggccct 300 gggaccaaag tggatatcaa a 321 <210> 88 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 88 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Ser Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Ile Leu Thr Val Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Asn Ser Asn Pro Phe 85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 100 105 <210> 89 <211> 381 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 89 gaggtgcagc tggtgcagtc tgggggagcc tggtacagc ctggggggtc cctgagactc 60 tcctgtacag cctctggttt caccttcagt aactacgaca tgcactggt ccgccaact 120 acaggaaaag gtctggagtg gatctcagct attgatactg ctggtgacac attackatcca 180 ggctccgtga agggccgatt caccgtctcc agaaaatg ccagaactc cttttatctt 240 caaatgaaca gcctgagagc cggggacacg gctgtgtatt actgtgcaag ggaggggaag 300 tattacgata ttttgactgg tgactaccac tactacggta tggacgtctg gggccaaggg 360 accacggtca ccgtctcctc a 381 <210> 90 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 90 Glu Val Gln Leu Val Gln Ser Gly Gly Ala Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Asp Met His Trp Val Arg Gln Thr Thr Gly Lys Gly Leu Glu Trp Ile 35 40 45 Ser Ala Ile Asp Thr Ala Gly Asp Thr Tyr Tyr Pro Gly Ser Val Lys 50 55 60 Gly Arg Phe Thr Val Ser Arg Glu Asn Ala Lys Asn Ser Phe Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Glu Gly Lys Tyr Tyr Asp Ile Leu Thr Gly Asp Tyr His Tyr Tyr 100 105 110 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 91 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 91 ggtttcacct tcagtaacta cgac 24 <210> 92 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 92 Gly Phe Thr Phe Ser Asn Tyr Asp 1 5 <210> 93 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 93 attgatactg ctggtgacac a 21 <210> 94 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 94 Ile Asp Thr Ala Gly Asp Thr 1 5 <210> 95 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 95 gcaagggagg ggaagtatta cgatattttg actggtgact accactacta cggtatggac 60 gtc 63 <210> 96 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 96 Ala Arg Glu Gly Lys Tyr Tyr Asp Ile Leu Thr Gly Asp Tyr His Tyr 1 5 10 15 Tyr Gly Met Asp Val 20 <210> 97 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 97 gccatccgga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgtc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 120 gggaaagcccc ctaagcgact gatctatgct acatccagtt tgcaagtgg ggtcccatca 180 aggttcagcg gcagtggatc tgggacagaa ttcactca caatcagcag cctgcagcct 240 gaagattttg caacttatta ctgtctacag catatagtt acccgctcac tttcggcgga 300 324 <210> 98 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 98 Wing Ile Arg Met Thr Gln To Be Pro To Be Leu To Be Val Gly 1 5 10 15 Asp Arg Val Thr and Cys Arg Only Serve as Gln Gly and Arg Asp 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Ala Thr Ser Leu Ser Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 99 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 99 cagggcatta gaaatgat 18 <210> 100 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 100 Gln Gly Ile Arg Asn Asp 1 5 <210> 101 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 101 gctacatcc 9 <210> 102 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 102 Ala Thr Ser 1 <210> 103 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 103 ctacagcata atagttaccc gctcact 27 <210> 104 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 104 Leu Gln His Asn Ser Tyr Pro Leu Thr 1 5 <210> 105 <211> 378 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 105 gaggtgcagc tggtggagtc tgggggagcc ttggtacagc ctggggggtc cctgagactc 60 tcctgtacag cctctggttt caccttcagt aactacgaca tgcactgggt ccgccaaact 120 acaggaaaag gtctggagtg gatctcagct attgatactg ctggtgacac atactatcca 180 ggctccgtga aggggccgatt caccgtctcc agagaaaatg ccaagaactc cttttatctt 240 caaatgaaca gcctgagagc cggggacacg gctgtgtatt actgtgcaag ggaggggaag 300 tattacgata ttttgactgg tgactaccac tactacggta tggacgtctg gggccaaggg 360 accacggtca ccgtctcc 378 <210> 106 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 106 Glu Val Gln Leu Val Glu Ser Gly Gly Ala Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Asp Met His Trp Val Arg Gln Thr Thr Gly Lys Gly Leu Glu Trp Ile 35 40 45 Ser Ala Ile Asp Thr Ala Gly Asp Thr Tyr Tyr Pro Gly Ser Val Lys 50 55 60 Gly Arg Phe Thr Val Ser Arg Glu Asn Ala Lys Asn Ser Phe Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Glu Gly Lys Tyr Tyr Asp Ile Leu Thr Gly Asp Tyr His Tyr Tyr 100 105 110 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115 120 125 <210> 107 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 107 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgtc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 120 gggaaagccc ctaagcgact gatctatgct acatccagtt tgcaaagtgg ggtcccatca 180 aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240 gaagattttg caacttatta ctgtctacag cataatagtt acccgctcac tttcggcgga 300 gggaccaagc tggagatcaa a 321 <210> 108 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 108 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Ala Thr Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 109 <211> 372 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 109 caggtgcagc tggtgcagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cgtctgggtt cacctttagt aactttggca tgcactgggt ccgccaggct 120 ccaggcaagg ggctggagtg ggtggcagtt atatggtttg atgaaattga taatactat 180 gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240 ccgcaaatga acagcctgag agccgaagac acggctgtgt attactgtgc gcgagaagat 300 tacgatattt tgactggtta ctattacgct atggacgtct ggggccaagg gaccacggtc 360 accgtctcct ca 372 <210> 110 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 110 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Phe Asp Glu Ile Asp Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Pro Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Asp Tyr Asp Ile Leu Thr Gly Tyr Tyr Tyr Ala Met Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 111 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 111 gggttcacct ttagtaactt tggc 24 <210> 112 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 112 Gly Phe Thr Phe Ser Asn Phe Gly 1 5 <210> 113 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 113 atatggtttg atgaaattga taaa 24 <210> 114 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 114 Ile Trp Phe Asp Glu Ile Asp Lys 1 5 <210> 115 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 115 gcgcgagaag attacgatat tttgactggt tactattacg ctatggacgt c 51 <210> 116 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 116 Ala Arg Glu Asp Tyr Asp Ile Leu Thr Gly Tyr Tyr Tyr Ala Met Asp 1 5 10 15 Val <210> 117 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 117 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 120 gggaaagccc ctaagcgcct aatctatgct gcatcccgtt tgcaaagtgg ggtcccatcg 180 aggttcagcg gcagtggatc tgggacagaa ttcactctca caatcagcag cctgcagcct 240 gaagattttg gaacttatta ctgtctacag cataatagtc accccacctt cggccaaggg 300 accaaggtgg agatcaaacg a 321 <210> 118 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 118 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Thr Tyr Tyr Cys Leu Gln His Asn Ser His Pro Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 119 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 119 cagggcatta gaaatgat 18 <210> 120 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 120 Gln Gly Ile Arg Asn Asp 1 5 <210> 121 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 121 gctgcatcc 9 <210> 122 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 122 Ala Ala Ser 1 <210> 123 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 123 ctacagcata atagtcaccc cacc 24 <210> 124 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 124 Leu Gln His Asn Ser His Pro Thr 1 5 <210> 125 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 125 caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cgtctgggtt cacctttagt aactttggca tgcactgggt ccgccaggct 120 ccaggcaagg ggctggagtg ggtggcagtt atatggtttg atgaaattga taatactat 180 gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240 ccgcaaatga acagcctgag agccgaagac acggctgtgt attactgtgc gcgagaagat 300 tacgatattt tgactggtta ctattacgct atggacgtct ggggccaagg gaccacggtc 360 accgtctcc 369 <210> 126 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 126 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Phe Asp Glu Ile Asp Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Arg Asp Asn Serves Lys Asn Thr Leu Tyr 65 70 75 80 Pro Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Only Arg Glu Asp Tyr Asp With Thr Gly Tyr Tyr Tyr Ala Met Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115 120 <210> 127 <211> 318 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 127 gataccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcaagtca gggcattaga aatgatttag gctggtatca gcagaaacca 120 gggaaagcccc ctaagcgcct aatctatgct gcatcccgtt tgcaagtgg ggtcccatcg 180 aggttcagcg gcagtggatc tgggacagaa ttcactca caatcagcag cctgcagcct 240 gaagattttg gaacttatta ctgtctacag cataatagtc accccacctt cggccaaggg 300 accaaggtgg agatcaaa 318 <210> 128 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 128 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Lys Asn Asp 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Thr Tyr Tyr Cys Leu Gln His Asn Ser His Pro Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 129 <211> 381 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 129 gaggtgcagc tggtggagtc ggggggaggc atggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctccagt aactacgaca tgcactgggt ccgccaagct 120 acaggaaaag gtctggagtg ggtctcaagt attgatactg ctggggacac ttactatcca 180 gactccgtga agggccgctt tatcatctcc agagaaaatg ccaaaaactc cctgtatctt 240 caaatgaata gcctgagagc cggggacacg gctgtgtatt actgtacaag ggagccccga 300 aattacgaaa ttttgactgg tcactaccac taccacggta tggacatctg gggccaaggg 360 accacggtca ccgtctcctc a 381 <210> 130 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 130 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Met Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ser Ser Asn Tyr 20 25 30 Asp Met His Trp Val Arg Gln Ala Thr Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Asp Thr Ala Gly Asp Thr Tyr Tyr Pro Asp Ser Val Lys 50 55 60 Gly Arg Phe Ile Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys Thr 85 90 95 Arg Glu Pro Arg Asn Tyr Glu Ile Leu Thr Gly His Tyr His Tyr His 100 105 110 Gly Met Asp Ile Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 131 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 131 ggattcacct ccagtaacta cgac 24 <210> 132 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 132 Gly Phe Thr Ser Ser Asn Tyr Asp 1 5 <210> 133 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 133 attgatactg ctggggacac t 21 <210> 134 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 134 Ile Asp Thr Ala Gly Asp Thr 1 5 <210> 135 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 135 acaagggagc cccgaaatta cgaaattttg actggtcact accactacca cggtatggac 60 atc 63 <210> 136 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 136 Thr Arg Glu Pro Arg Asn Tyr Glu Ile Leu Thr Gly His Tyr His Tyr 1 5 10 15 His Gly Met Asp Ile 20 <210> 137 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 137 gacatccaga tgacccagtc gccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcaagtca ggccattaga aatgatttag gctggtatca gcagaaacca 120 gggaaagccc ctaaactcct gatctatact gcattcagtt tacagagtgg ggtcccatca 180 aggttcagcg gcagtaaatc tggcacagac ttcactctca ccatcagcag cctgcagcct 240 gaagattttg cgacttatta ctgtctgcag gattacacta atcctcggac gttcggccaa 300 gggaccaagg tggagatcaa acga 324 <210> 138 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 138 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ala Ile Arg Asn Asp 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Thr Ala Phe Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Lys Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Asp Tyr Thr Asn Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 139 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 139 caggccatta gaaatgat 18 <210> 140 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 140 Gln Ala Ile Arg Asn Asp 1 5 <210> 141 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 141 actgcattc 9 <210> 142 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 142 Thr Ala Phe 1 <210> 143 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 143 ctgcaggatt acactaatcc tcggacg 27 <210> 144 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 144 Leu Gln Asp Tyr Thr Asn Pro Arg Thr 1 5 <210> 145 <211> 378 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 145 gaggtgcagc tggtggagtc ggggggaggc atggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctccagt aactacgaca tgcactgggt ccgccaagct 120 acaggaaaag gtctggagtg ggtctcaagt attgatactg ctggggacac ttactatcca 180 gactccgtga agggccgctt tatcatctcc agagaaaatg ccaaaaactc cctgtatctt 240 caaatgaata gcctgagagc cggggacacg gctgtgtatt actgtacaag ggagccccga 300 aattacgaaa ttttgactgg tcactaccac taccacggta tggacatctg gggccaaggg 360 accacggtca ccgtctcc 378 <210> 146 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 146 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Met Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ser Ser Asn Tyr 20 25 30 Asp Met His Trp Val Arg Gln Ala Thr Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Asp Thr Ala Gly Asp Thr Tyr Tyr Pro Asp Ser Val Lys 50 55 60 Gly Arg Phe Ile Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Gly Asp Thr Ala Val Tyr Tyr Cys Thr 85 90 95 Arg Glu Pro Arg Asn Tyr Glu Ile Leu Thr Gly His Tyr His Tyr His 100 105 110 Gly Met Asp Ile Trp Gly Gln Gly Thr Thr Val Thr Val Ser 115 120 125 <210> 147 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 147 gccatccaga tgacccagtc gccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcaagtca ggccattaga aatgatttag gctggtatca gcagaaacca 120 gggaaagcccc ctaaactccct gatctatact gcattcagtt tacagagtgg ggtcccatca 180 aggttcagcg gcagtaaatc tgcacagac ttcactca ccatcagcag cctgcagcct 240 gaagattttg cgacttatta ctgtctgcag gattacacta atcctcggac gttcggccaa 300 321 - slowly slowly <210> 148 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 148 Wing Ile Gln Met Thr Gln To Be Pro To Be Leu To Wing To Be Val Gly 1 5 10 15 Asp Arg Val Thr With Thr Cys Arg Only Serves On Gln With Arg Asn Asp 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Ile 35 40 45 Tyr Thr Ala Phe Ser Leu Gln Ser Gly Val Pro Ser Arg PHE Ser Gly 50 55 60 Ser Lys Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Asp Tyr Thr Asn Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 149 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 149 caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cctctggatt cgccttcagt aactatggca tgcactgggt ccgccaggct 120 ccaggcaagg ggctggaatg ggtgacattt atatcatatg atggaagtaa taatactat 180 gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaagtga acagcctgag agctgaggac acggctgtgt attactgtgc gaaagaagca 300 gtatgctg ccctctttga ctactggggc cagggaaccc tggtcaccgt ctcctca 357 <210> 150 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 150 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Asn Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Thr Phe Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Val Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Ala Val Leu Ala Ala Leu Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 151 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 151 ggattcgcct tcagtaacta tggc 24 <210> 152 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 152 Gly Phe Ala Phe Ser Asn Tyr Gly 1 5 <210> 153 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 153 atatcatatg atggaagtaa taaa 24 <210> 154 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 154 Ile Ser Tyr Asp Gly Ser Asn Lys 1 5 <210> 155 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 155 gcgaaagaag cagtattagc tgccctcttt gactac 36 <210> 156 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 156 Wing Lys Glu Wing Val Leu Wing Wing Leu Phe Asp Tyr 1 5 10 <210> 157 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 157 gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60 atcaactgca agtccagcca gagtgtttta tacagctcca acaatcagaa ctacttagct 120 tggtaccagc agaaaccagg acagcctcct aagctgctca tttactgggc atctacccgg 180 gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240 atcaacagcc tgcaggctga agatgtggca gttattact gtcagcaata ttatagtact 300 cctacgtcg gccaagggac caaggtggaa atcaaa 336 <210> 158 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 158 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30 Ser Asn Asn Gln Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Asn Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Thr Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 159 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 159 cagagtgttt tatacagctc caacaatcag aactac 36 <210> 160 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 160 Gln Ser Val Leu Tyr Ser Ser Donkey Donkey Gln Donkey Tyr 1 5 10 <210> 161 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 161 tgggcatct 9 <210> 162 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 162 Trp Ala Dear 1 <210> 163 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 163 cagcaatatt atagtactcc tacg 24 <210> 164 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 164 Gln Gln Tyr Tyr Ser Thr Pro Thr 1 5 <210> 165 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 165 caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cctctggatt cacgttcaat acctatggca tgcactgggt ccgccaggct 120 ccagtcaagg ggctggagtg ggtggcattt atatcaaatg ataagagtaa tacattctat 180 gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgttt 240 ctggaaatga acagcctgac agctgaggac acggctgttt attactgtgc gaaagagtcc 300 attttagcag ccctctttga ctactggggc cagggaaccc tggtcactgt ctcctca 357 <210> 166 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 166 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Val Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Ser Asn Asp Lys Ser Asn Thr Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Glu Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Ser Ile Leu Ala Ala Leu Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 167 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 167 ggattcacgt tcaataccta tggc 24 <210> 168 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 168 Gly Phe Thr Phe Asn Thr Tyr Gly 1 5 <210> 169 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 169 atatcaaatg ataagagtaa taca 24 <210> 170 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 170 Ile Ser Asn Asp Lys Ser Asn Thr 1 5 <210> 171 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 171 gcgaaagagt ccattttagc agccctcttt gactac 36 <210> 172 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 172 Ala Ala Lys Glu Ser Ile Leu Ala Ala Leu Phe Asp Tyr 1 5 10 <210> 173 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 173 gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60 atcaactgca agtccagcca gagtgtttta tacagctcca acaataagaa ttacttagct 120 tggtaccaac agaaaccaag acagcctctt aaactactca tttactgggc atctattcgg 180 gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240 atcagcagcc tgcaggctga agatgtggca gttattact gtcagcaatt ttatagtgtt 300 cccactttg gccaggggac caagctggag atcaaa 336 <210> 174 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 174 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30 Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Arg Gln 35 40 45 Pro Leu Lys Leu Leu Ile Tyr Trp Ala Ser Ile Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Phe Tyr Ser Val Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 175 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 175 cagagtgttt tatacagctc caacaataag aattac 36 <210> 176 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 176 Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr 1 5 10 <210> 177 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 177 tgggcatct 9 <210> 178 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 178 Trp Ala Dear 1 <210> 179 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 179 cagcaatttt atagtgttcc cact 24 <210> 180 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 180 Gln Gln Phe Tyr Ser Val Pro Thr 1 5 <210> 181 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 181 caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cctctggatt cacgtttagt acctttggca tgcactgggt ccgccaggct 120 ccagtcaagg ggctggagtg ggtggctttt atatcaaatg ataagaataa taaattctat 180 gcagactccg tgaagggccg attcaccatc tccagagaca attccaggga cacgctatat 240 ctgcaaatga acagcctgac acctgaggac acggctgttt attactgtgc gaaagagtcc 300 attttagcag ccctctttga ctactggggc cagggaaccc tggtcaccgt ctcctca 357 <210> 182 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 182 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Phe 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Val Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Ser Asn Asp Lys Asn Asn Lys Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Asp Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Ser Ile Leu Ala Ala Leu Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 183 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 183 ggattcacgt ttagtacctt tggc 24 <210> 184 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 184 Gly Phe Thr Phe Ser Thr Phe Gly 1 5 <210> 185 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 185 atatcaaatg ataagaataa taaa 24 <210> 186 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 186 Ile Ser Asn Asp Lys Asn Asn Lys 1 5 <210> 187 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 187 gcgaaagagt ccattttagc agccctcttt gactac 36 <210> 188 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 188 Ala Ala Lys Glu Ser Ile Leu Ala Ala Leu Phe Asp Tyr 1 5 10 <210> 189 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 189 gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60 atcaactgca agtccagcca gagtgtttta tacagctcca acaataaaaa ttacttagct 120 tggtaccagc agaaaccagg acagcctctt aaacttctca tttactgggc atctattcgg 180 gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240 atcagcagcc tgcaggctga agatgtggca gttattact gtcagcaatt ttatactgtt 300 cccactttg gcctggggac caagctggag atcaaa 336 <210> 190 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 190 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30 Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Leu Lys Leu Leu Ile Tyr Trp Ala Ser Ile Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Phe Tyr Thr Val Pro Thr Phe Gly Leu Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 191 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 191 cagagtgttt tatacagctc caacaataaa aattac 36 <210> 192 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 192 Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr 1 5 10 <210> 193 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 193 tgggcatct 9 <210> 194 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 194 Trp Ala Dear 1 <210> 195 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 195 cagcaatttt atactgttcc cact 24 <210> 196 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 196 Gln Gln Phe Tyr Thr Val Pro Thr 1 5 <210> 197 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 197 caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgtag cctctggatt caccttcagg aactatgaca tgcactgggt ccgccaggct 120 cctggcaagg ggctggaatg ggtggcagtt acatcatctg atggacttaa taaattctat 180 tcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtct 240 ctgcaaatta ccggcctgag agctgaggac acggctgtgt attactgtgc gaaagagtcc 300 attttagcag ccctctttga ctactggggc cagggaaccc tggtcaccgt ctcctca 357 <210> 198 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 198 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Arg Asn Tyr 20 25 30 Asp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Thr Ser Ser Asp Gly Leu Asn Lys Phe Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Ser 65 70 75 80 Leu Gln Ile Thr Gly Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Ser Ile Leu Ala Ala Leu Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 199 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 199 ggattcacct tcaggaacta tgac 24 <210> 200 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 200 Gly Phe Thr Phe Arg Asn Tyr Asp 1 5 <210> 201 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 201 acatcatctg atggacttaa taaa 24 <210> 202 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 202 Thr Ser Ser Asp Gly Leu Asn Lys 1 5 <210> 203 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 203 gcgaaagagt ccattttagc agccctcttt gactac 36 <210> 204 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 204 Ala Ala Lys Glu Ser Ile Leu Ala Ala Leu Phe Asp Tyr 1 5 10 <210> 205 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 205 gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60 atcaactgca agtccagcca gagtgtttta tacagctcca acaataagaa ctacttggct 120 tggtaccagc agaaaccagg acagcctcct aagctgctct tttactgggc atctacccgg 180 gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240 atcagcagcc tgcaggctga agatgtggca gttattact gtcagcaaca ttatactact 300 cccactttg gccaggggac caagctggag atcaaa 336 <210> 206 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 206 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30 Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Phe Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 His Tyr Thr Thr Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 207 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 207 cagagtgttt tatacagctc caacaataag aactac 36 <210> 208 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 208 Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr 1 5 10 <210> 209 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 209 tgggcatct 9 <210> 210 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 210 Trp Ala Dear 1 <210> 211 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 211 cagcaacatt atactactcc cact 24 <210> 212 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 212 Gln Gln His Tyr Thr Thr Pro Thr 1 5 <210> 213 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 213 caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt agctatggca tgcactgggt ccgccagact 120 ccgggcaagg ggctggagtg ggtggcattt atatcatatg atggaaataa taaatactat 180 gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgttt 240 ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtgc gaaagagtcc 300 attttagcag ccctctttga ctactggggc cagggaaccc tggtcaccgt ctcctca 357 <210> 214 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 214 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Ser Ile Leu Ala Ala Leu Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 215 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 215 ggattcacct tcagtagcta tggc 24 <210> 216 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 216 Gly Phe Thr Phe Ser Ser Tyr Gly 1 5 <210> 217 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 217 atatcatatg atggaaataa taaa 24 <210> 218 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 218 Ile Ser Tyr Asp Gly Asn Asn Lys 1 5 <210> 219 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 219 gcgaaagagt ccattttagc agccctcttt gactac 36 <210> 220 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 220 Ala Ala Lys Glu Ser Ile Leu Ala Ala Leu Phe Asp Tyr 1 5 10 <210> 221 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 221 gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60 atcaactgca agtccagcca gagtgtttta tacagctcca acaataagaa ctacttagct 120 tggtaccagc agaaacctgg acagcctcct aagctgctca tttactgggc atctacccgg 180 gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240 atcagcagcc tgcaggctga agatgtggca ctttattact gtcaacaata ttataatact 300 cccactttg gccaggggac caagctggag atcaaa 336 <210> 222 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 222 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30 Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Leu Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Asn Thr Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 223 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 223 cagagtgttt tatacagctc caacaataag aactac 36 <210> 224 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 224 Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr 1 5 10 <210> 225 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 225 tgggcatct 9 <210> 226 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 226 Trp Ala Dear 1 <210> 227 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 227 caacaatatt ataatactcc cact 24 <210> 228 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 228 Gln Gln Tyr Tyr Asn Thr Pro Thr 1 5 <210> 229 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 229 caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt agctatggca tgcactgggt ccgccaggct 120 ccagtcaagg ggctggagtg ggtggcattt atatcatatg atggaagtaa taaatactat 180 gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240 ctccaaatga acagcctgac agctgaggac acggctgttt attactgtgc gaaagagtcc 300 attttagcag ccctctttga ctactggggc cagggaaccc tggtcaccgt ctcctca 357 <210> 230 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 230 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Val Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Ser Ile Leu Ala Ala Leu Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 231 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 231 ggattcacct tcagtagcta tggc 24 <210> 232 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 232 Gly Phe Thr Phe Ser Ser Tyr Gly 1 5 <210> 233 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 233 atatcatatg atggaagtaa taaa 24 <210> 234 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 234 Ile Ser Tyr Asp Gly Ser Asn Lys 1 5 <210> 235 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 235 gcgaaagagt ccattttagc agccctcttt gactac 36 <210> 236 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 236 Ala Ala Lys Glu Ser Ile Leu Ala Ala Leu Phe Asp Tyr 1 5 10 <210> 237 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 237 gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60 atcaactgca agtccagcca gagtgtttta tacagttcca acaataagaa ctacttagct 120 tggtaccagc agaaaccaag acagcctcct aagctgctca tttactgggc atctattcgg 180 gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cattctcacc 240 atcagcagcc tgcaggctga agatgtggca gttattact gtcagcaatt ttatagtatt 300 cccactttg gccaggggac caagctggag atcaaa 336 <210> 238 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 238 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30 Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Arg Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ile Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ile Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Phe Tyr Ser Ile Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 239 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 239 cagagtgttt tatacagttc caacaataag aactac 36 <210> 240 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 240 Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr 1 5 10 <210> 241 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 241 tgggcatct 9 <210> 242 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 242 Trp Ala Dear 1 <210> 243 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 243 cagcaatttt atagtattcc cact 24 <210> 244 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 244 Gln Gln Phe Tyr Ser Ile Pro Thr 1 5 <210> 245 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 245 caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagt agctatggca tgcactgggt ccgccaggct 120 ccagtcaagg ggctggagtg ggtggcattt atatcaaatg ataaaagtaa taaattat 180 gcagactcct tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgac agctgaagac acggctgttt attactgtgc gaaagagtcc 300 attttagcag ccctctttga ctattggggc cagggaaccc tggtcaccgt ctcctca 357 <210> 246 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 246 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Val Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Ser Asn Asp Lys Ser Asn Lys Tyr Tyr Ala Asp Ser Leu 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Ser Ile Leu Ala Ala Leu Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 247 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 247 ggattcacct ttagtagcta tggc 24 <210> 248 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 248 Gly Phe Thr Phe Ser Ser Tyr Gly 1 5 <210> 249 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 249 atatcaaatg ataaaagtaa taaa 24 <210> 250 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 250 Ile Ser Asn Asp Lys Ser Asn Lys 1 5 <210> 251 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 251 gcgaaagagt ccattttagc agccctcttt gactat 36 <210> 252 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 252 Ala Ala Lys Glu Ser Ile Leu Ala Ala Leu Phe Asp Tyr 1 5 10 <210> 253 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 253 gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60 atcaactgca agtccagcca gagtgtttta tacagctcca acaataagaa ctacttagct 120 tggtaccagc agaaaccaag acagcctcct aagctactca tttactgggc atctattcgg 180 gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240 atcagcagcc tgcaggctga agatgtggca gttattact gtcaacaatt ttatagtgtt 300 cccactttg gccaggggac caagctggag atcaaa 336 <210> 254 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 254 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30 Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Arg Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ile Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Phe Tyr Ser Val Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 255 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 255 cagagtgttt tatacagctc caacaataag aactac 36 <210> 256 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 256 Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr 1 5 10 <210> 257 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 257 tgggcatct 9 <210> 258 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 258 Trp Ala Dear 1 <210> 259 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 259 caacaatttt atagtgttcc cact 24 <210> 260 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 260 Gln Gln Phe Tyr Ser Val Pro Thr 1 5 <210> 261 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 261 caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt agctatggca tgcactgggt ccgccaggct 120 ccagtcaagg ggctggagtg ggtggcattt atatcatttg atggaagtaa taaatactat 180 acagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240 ctccaaatga acagcctgac agctgaggac acggctattt attactgtgc gaaagagtcc 300 attttagcag ccctctttga ctactggggc cagggaaccc tggtcactgt ctcctca 357 <210> 262 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 262 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Val Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Ser Phe Asp Gly Ser Asn Lys Tyr Tyr Thr Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Lys Glu Ser Ile Leu Ala Ala Leu Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 263 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 263 ggattcacct tcagtagcta tggc 24 <210> 264 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 264 Gly Phe Thr Phe Ser Ser Tyr Gly 1 5 <210> 265 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 265 atatcatttg atggaagtaa taaa 24 <210> 266 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 266 Ile Ser Phe Asp Gly Ser Asn Lys 1 5 <210> 267 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 267 gcgaaagagt ccattttagc agccctcttt gactac 36 <210> 268 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 268 Ala Ala Lys Glu Ser Ile Leu Ala Ala Leu Phe Asp Tyr 1 5 10 <210> 269 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 269 gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60 atcaactgca agtccagcca gagtgtttta tacagctcca acaataagaa ctacttagct 120 tggtaccagc agaaaccaag acagcctcct aacctgctca tttactgggc atctattcgg 180 gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240 atcagcagcc tgcaggctga agatgtggca ttttattact gtcagcaatt ttatagtatt 300 cccactttg gccaggggac caagctggag atcaaa 336 <210> 270 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 270 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30 Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Arg Gln 35 40 45 Pro Pro Asn Leu Leu Ile Tyr Trp Ala Ser Ile Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Phe Tyr Tyr Cys Gln Gln 85 90 95 Phe Tyr Ser Ile Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 271 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 271 cagagtgttt tatacagctc caacaataag aactac 36 <210> 272 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 272 Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr 1 5 10 <210> 273 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 273 tgggcatct 9 <210> 274 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 274 Trp Ala Dear 1 <210> 275 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 275 cagcaatttt atagtattcc cact 24 <210> 276 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 276 Gln Gln Phe Tyr Ser Ile Pro Thr 1 5 <210> 277 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 277 caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagg acctatggca tgcactgggt ccgccaggct 120 ccagtcaagg ggctggagtg ggtggcattt atatcaaagg atggaagtga taatactat 180 gtagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgttt 240 ctgcaaatga acagcctgac agctgaggac acggctgttt attattgtgc gaaagagtcc 300 attttagcag ccctctttga ctactggggc cagggaaccc tggtcactgt ctcctca 357 <210> 278 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 278 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Thr Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Val Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Ser Lys Asp Gly Ser Asp Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Ser Ile Leu Ala Ala Leu Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 279 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 279 ggattcacct ttaggaccta tggc 24 <210> 280 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 280 Gly Phe Thr Phe Arg Thr Tyr Gly 1 5 <210> 281 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 281 atatcaaagg atggaagtga taaa 24 <210> 282 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 282 Ile Ser Lys Asp Gly Ser Asp Lys 1 5 <210> 283 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 283 gcgaaagagt ccattttagc agccctcttt gactac 36 <210> 284 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 284 Ala Ala Lys Glu Ser Ile Leu Ala Ala Leu Phe Asp Tyr 1 5 10 <210> 285 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 285 gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60 atcaactgca agtccagcca gagtgtttta tacagctcca acaataagaa ctacttagct 120 tggtaccagc agaaaccaag acagcctcct aaactcctca tttactgggc atctaatcgg 180 gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240 atcagcagcc tgcaggctga agatgtggca gttattact gtcagcaatt ttatagtgtt 300 cccactttg gccaggggac caagctggag atcaaa 336 <210> 286 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 286 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30 Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Arg Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Asn Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Phe Tyr Ser Val Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 287 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 287 cagagtgttt tatacagctc caacaataag aactac 36 <210> 288 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 288 Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr 1 5 10 <210> 289 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 289 tgggcatct 9 <210> 290 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 290 Trp Ala Dear 1 <210> 291 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 291 cagcaatttt atagtgttcc cact 24 <210> 292 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 292 Gln Gln Phe Tyr Ser Val Pro Thr 1 5 <210> 293 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 293 caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagt agctatggca tgcactgggt ccgccaggct 120 ccagtcaagg ggctggagtg ggtggcattt atatcaaatg ataaaagtaa taaatactat 180 gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgac agctgaggac acggctgttt attactgtgc gaaagagtcc 300 attttagcag ccctctttga ctcctggggc cagggaaccc tggtcaccgt ctcctca 357 <210> 294 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 294 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Val Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Ser Asn Asp Lys Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Glu Ser Ile Leu Ala Ala Leu Phe Asp Ser Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 295 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 295 ggattcacct ttagtagcta tggc 24 <210> 296 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 296 Gly Phe Thr Phe Ser Ser Tyr Gly 1 5 <210> 297 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 297 atatcaaatg ataaaagtaa taaa 24 <210> 298 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 298 Ile Ser Asn Asp Lys Ser Asn Lys 1 5 <210> 299 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 299 gcgaaagagt ccattttagc agccctcttt gactcc 36 <210> 300 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 300 Ala Ala Lys Glu Ser Ile Leu Ala Ala Leu Phe Asp Ser 1 5 10 <210> 301 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 301 gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60 atcaactgca agtccagcca gagtgtttta tacagctcca acaataagaa ctacttagct 120 tggtaccagc agaaaccaag acagcctcct aagctgctca tttactgggc atctattcgg 180 gaatccgggg tccctgaccg attcagtggc agcgggtctg gggcagattt cactctcacc 240 atcagcagcc tgcaggctgc agatgtggca gttattact gtcagcaatt ttatagtgtt 300 cccactttg gccaggggac caagctggag atcaaa 336 <210> 302 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 302 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30 Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Arg Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ile Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Ala Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Ala Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Phe Tyr Ser Val Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 303 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 303 cagagtgttt tatacagctc caacaataag aactac 36 <210> 304 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 304 Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr 1 5 10 <210> 305 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 305 tgggcatct 9 <210> 306 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 306 Trp Ala Dear 1 <210> 307 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic <400> 307 cagcaatttt atagtgttcc cact 24 <210> 308 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 308 Gln Gln Phe Tyr Ser Val Pro Thr 1 5

Claims

1. A pharmaceutical agent for treating hyperammonemia in a subject in need thereof, comprising a glucagon signaling pathway antagonist, The method is characterized in that it is used for administering to a subject so as to alleviate or reduce the severity of at least one symptom or complication associated with hyperammonemia, The hyperammonemia is associated with a deficiency in one or more urea cycle enzymes selected from the group consisting of carbamyl phosphate synthetase (CPS1), N-acetylglutamate synthetase (NAGS), ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL), and arginase (AR1), or is associated with glutamine synthase deficiency. The medicine.

2. The pharmaceutical described in claim 1, wherein the hyperammonemia is acquired.

3. The pharmaceutical described in claim 1, wherein the hyperammonemia is congenital hyperammonemia.

4. The glucagon signaling pathway antagonist is an isolated human monoclonal antibody or antigen-binding fragment thereof that specifically binds to the glucagon receptor; The isolated human monoclonal antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) within a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 34, and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) within a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO:

42. The pharmaceutical composition according to any one of claims 1 to 3.

5. The glucagon signaling pathway antagonist, wherein the glucagon signaling pathway antagonist is an isolated human monoclonal antibody or antigen-binding fragment thereof that specifically binds to the glucagon receptor; The isolated human monoclonal antibody or antigen-binding fragment thereof comprises an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 36, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 46, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:

48. The pharmaceutical composition according to any one of claims 1 to 4.

6. The glucagon signaling pathway antagonist, wherein the glucagon signaling pathway antagonist is an isolated human monoclonal antibody or antigen-binding fragment thereof that specifically binds to the glucagon receptor; The isolated human monoclonal antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 34 and an LCVR comprising the amino acid sequence set forth in SEQ ID NO:

42. The pharmaceutical composition according to any one of claims 1 to 5.

7. A pharmaceutical described in any one of claims 1 to 6, characterized in that it is used in combination with at least one additional therapeutic agent or supplement. (i) one or more amino acid preparations selected from Cyclinex, EAA, UCD-I, UCD-II, and individual branched-chain amino acids; (ii) antioxidants and / or electrolytes; (iii) L-citrulline and / or L-arginine free base; (iv) hemodialysis and / or continuous renal replacement; and / or (v) non-absorbable antibiotics, rifaximin, lactulose, sodium phenylbutyrate, sodium benzoate, sodium phenylacetate, glycerol phenylbutyrate, carbamyl glutamate, a second GCG inhibitor, and / or a second GCGR antagonist The pharmaceutical composition according to any one of claims 1 to 7, which is used in combination with

9. A pharmaceutical comprising a glucagon signaling pathway antagonist for treating a urea cycle disorder in a subject exhibiting elevated levels of ammonia.

10. The subject having a urea cycle disorder (i) a deficiency in one or more urea cycle enzymes selected from the group consisting of carbamyl phosphate synthetase (CPS1), N-acetylglutamate synthetase (NAGS), ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL), and arginase (AR1); and / or (ii) a deficiency in one or more urea cycle transporters selected from ornithine translocase (ORNT1) and citrin The pharmaceutical composition according to claim 9, wherein the patient is suffering from 11. The glucagon signaling pathway antagonist, wherein the glucagon signaling pathway antagonist is an isolated human monoclonal antibody or antigen-binding fragment thereof that specifically binds to the glucagon receptor; The isolated human monoclonal antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) within a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 34, and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) within a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO:

42. The pharmaceutical composition according to claim 9 or 10.

12. The glucagon signaling pathway antagonist, wherein the glucagon signaling pathway antagonist is an isolated human monoclonal antibody or antigen-binding fragment thereof that specifically binds to the glucagon receptor; The isolated human monoclonal antibody or antigen-binding fragment thereof comprises an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 36, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 46, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:

48. The pharmaceutical composition according to any one of claims 9 to 11.

13. The glucagon signaling pathway antagonist, wherein the glucagon signaling pathway antagonist is an isolated human monoclonal antibody or antigen-binding fragment thereof that specifically binds to the glucagon receptor; The isolated human monoclonal antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 34 and an LCVR comprising the amino acid sequence set forth in SEQ ID NO:

42. The pharmaceutical composition according to any one of claims 9 to 12.

14. A pharmaceutical described in any one of claims 9 to 13, characterized in that it is used in combination with at least one additional therapeutic agent or supplement. (i) one or more amino acid preparations selected from Cyclinex, EAA, UCD-I, UCD-II, and individual branched-chain amino acids; (ii) antioxidants and / or electrolytes; (iii) L-citrulline and / or L-arginine free base; (iv) hemodialysis and / or continuous renal replacement; and / or (v) non-absorbable antibiotics, rifaximin, lactulose, sodium phenylbutyrate, sodium benzoate, sodium phenylacetate, glycerol phenylbutyrate, carbamyl glutamate, a second GCG inhibitor, and / or a second GCGR antagonist The pharmaceutical composition according to any one of claims 9 to 14, which is used in combination with 16. A pharmaceutical composition for treating hyperammonemia in a subject, comprising a glucagon signaling pathway antagonist, comprising: the glucagon signaling pathway antagonist is administered to a subject in combination with sodium phenylbutyrate or sodium benzoate such that the amount and / or dose of sodium phenylbutyrate or sodium benzoate is reduced compared to the amount and / or dose of sodium phenylbutyrate or sodium benzoate administered to the subject without the glucagon signaling pathway antagonist; The hyperammonemia is associated with a deficiency in one or more urea cycle enzymes selected from the group consisting of carbamyl phosphate synthetase (CPS1), N-acetylglutamate synthetase (NAGS), ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL), and arginase (AR1), or is associated with glutamine synthase deficiency. The medicine.

17. The pharmaceutical described in claim 16, wherein the glucagon signaling pathway antagonist is administered simultaneously with sodium phenylbutyrate and / or sodium benzoate.

18. The glucagon signaling pathway antagonist, wherein the glucagon signaling pathway antagonist is an isolated human monoclonal antibody or antigen-binding fragment thereof that specifically binds to the glucagon receptor; The isolated human monoclonal antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) within a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 34, and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) within a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO:

42. The pharmaceutical composition according to claim 16 or 17.

19. The glucagon signaling pathway antagonist, wherein the glucagon signaling pathway antagonist is an isolated human monoclonal antibody or antigen-binding fragment thereof that specifically binds to the glucagon receptor; The isolated human monoclonal antibody or antigen-binding fragment thereof comprises an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 36, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 46, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:

48. The pharmaceutical composition according to any one of claims 16 to 18.

20. The glucagon signaling pathway antagonist, wherein the glucagon signaling pathway antagonist is an isolated human monoclonal antibody or antigen-binding fragment thereof that specifically binds to the glucagon receptor; The isolated human monoclonal antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 34 and an LCVR comprising the amino acid sequence set forth in SEQ ID NO:

42. The pharmaceutical composition according to any one of claims 16 to 19.

21. A pharmaceutical composition for reducing blood ammonia levels in a subject with hyperammonemia, comprising a glucagon signaling pathway antagonist, The hyperammonemia is associated with a deficiency in one or more urea cycle enzymes selected from the group consisting of carbamyl phosphate synthetase (CPS1), N-acetylglutamate synthetase (NAGS), ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL), and arginase (AR1), or is associated with glutamine synthase deficiency. The medicine.

22. The glucagon signaling pathway antagonist, wherein the glucagon signaling pathway antagonist is an isolated human monoclonal antibody or antigen-binding fragment thereof that specifically binds to the glucagon receptor; The isolated human monoclonal antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) within a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 34, and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) within a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO:

42. The pharmaceutical composition of claim 21.

23. The glucagon signaling pathway antagonist, wherein the glucagon signaling pathway antagonist is an isolated human monoclonal antibody or antigen-binding fragment thereof that specifically binds to the glucagon receptor; The isolated human monoclonal antibody or antigen-binding fragment thereof comprises an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 36, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 46, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:

48. The pharmaceutical composition according to claim 21 or 22.

24. The glucagon signaling pathway antagonist, wherein the glucagon signaling pathway antagonist is an isolated human monoclonal antibody or antigen-binding fragment thereof that specifically binds to the glucagon receptor; The isolated human monoclonal antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 34 and an LCVR comprising the amino acid sequence set forth in SEQ ID NO:

42. The pharmaceutical composition according to any one of claims 21 to 23.

25. A pharmaceutical comprising a glucagon signaling pathway antagonist for reducing excessive weight loss and / or lowering blood glucose in a subject with a urea cycle disorder.

26. The glucagon signaling pathway antagonist, wherein the glucagon signaling pathway antagonist is an isolated human monoclonal antibody or antigen-binding fragment thereof that specifically binds to the glucagon receptor; The isolated human monoclonal antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) within a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 34, and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) within a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO:

42. The pharmaceutical composition of claim 25.

27. The glucagon signaling pathway antagonist, wherein the glucagon signaling pathway antagonist is an isolated human monoclonal antibody or antigen-binding fragment thereof that specifically binds to the glucagon receptor; The isolated human monoclonal antibody or antigen-binding fragment thereof comprises an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 36, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 38, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 46, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:

48. The pharmaceutical composition according to claim 25 or 26.

28. The glucagon signaling pathway antagonist, wherein the glucagon signaling pathway antagonist is an isolated human monoclonal antibody or antigen-binding fragment thereof that specifically binds to the glucagon receptor; The isolated human monoclonal antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 34 and an LCVR comprising the amino acid sequence set forth in SEQ ID NO:

42. The pharmaceutical composition according to any one of claims 25 to 27.

29. A pharmaceutical described in any one of claims 25 to 28, wherein the subject is consuming a high-protein diet.

Images