Anti-tmprss6 antibodies and uses thereof

Abstract

The present invention provides antibodies and antigen-binding fragments thereof that bind to Transmembrane Serine Protease 6 (TMPRSS6) on the surface of a cell and increase hepcidin expression, as well as methods of using anti-TMPRSS6 antibodies and fragments to treat iron metabolism disorders.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 006,695, filed April 7, 2020, entitled “Anti-TMPRSS6 Antibodies and Uses Thereof,” and U.S. Provisional Application No. 63 / 158,265, filed March 8, 2021, entitled “Anti-TMPRSS6 Antibodies and Uses Thereof,” the entire contents of which are hereby incorporated by reference.

[0003] sequence list

[0004] This application contains a sequence list, which has been submitted electronically in ASCII format and is hereby incorporated by full reference. The ASCII copy was created on March 29, 2021, and is named 1121-101PCT_SL.txt with a size of 132,677 bytes. Technical Field

[0005] This invention relates to antibodies and antigen-binding fragments that bind to TMPRSS6, and to the use of antibodies and antigen-binding fragments that bind to TMPRSS6 to treat iron metabolism disorders. Background Technology

[0006] Type II transmembrane serine protease 6 (TMPRSS6) is encoded by the TMPRSS6 gene and is primarily expressed in the liver. The structure of TMPRSS6 includes a type II transmembrane domain, followed by a sea urchin spermatin, intestinal peptidase, and agglutinin (SEA) domain (a stalk region containing two complement factors, C1r / C1s), a sea urchin embryonic growth factor and bone morphogenetic protein (CUB) domain, three low-density lipoprotein receptor (LDLR) class A repeat sequences, and a C-terminal trypsin-like serine protease domain (Wang, C.-Y. et al., *Fronts in Pharmacology*, 2014.5:114). Synonyms for TMPRSS6 (EC3.4.21) include: interstitial protease-2; transmembrane protease serine 6; membrane-bound chimeric serine protease interstitial protease-2; and MT2.

[0007] TMPRSS6 plays a significant role in iron homeostasis by regulating the BMP-SMAD signaling pathway, which modulates the expression of hepcidin (a hormone that controls iron absorption and mobilization from storage). Hepcidin (also known as HAMP (hepcidin antimicrobial protein or peptide), encoded by HAMP in humans and non-human primates and Hamp in mice and rats) regulates systemic iron homeostasis by controlling the functional activity of the membrane iron transporter, the only iron efflux channel. Hepcidin can reduce plasma iron levels by binding to the membrane iron transporter and causing internalization and degradation of the complex, thereby preventing iron absorption in the small intestine and the release of stored iron. Long-term elevated hepcidin levels lead to systemic iron deficiency, while hepcidin deficiency leads to systemic iron overload.

[0008] TMPRSS6 negatively regulates hepcidin production via a transmembrane signaling pathway that is triggered by iron deficiency and inhibits HAMP activation (Du, X. et al., Science 2008, 320:1088-1092; Wang, C.-Y. et al., Frontiers in Pharmacology 2014, 5:114). Low blood iron levels trigger this pathway to reduce hepcidin production, allowing more iron from diet to be absorbed in the intestine and transported from storage sites into the bloodstream. In rats with acute iron deficiency, the hepatic TMPRSS6 protein content is upregulated, leading to inhibition of hepcidin expression and production (Wang, C.-Y. et al., Frontiers in Pharmacology 2014, 5:114). Mutations in the entire TMPRSS6 molecule (especially in the extracellular domain) have been identified in individuals with iron deficiency anemia, particularly iron-refractory iron deficiency anemia (IRIDA), which is unresponsive to oral iron therapy and only partially responsive to parenteral iron therapy (Wang, C.-Y. et al., Frontiers in Pharmacology 2014.5:114). Loss-of-function mutations in TMPRSS6 in humans lead to increased hepcidin levels and iron deficiency anemia (Camaschella, C., New England Journal of Medicine 2013.168:24), due to excessive hepcidin production resulting in insufficient iron absorption and utilization.

[0009] Iron overload syndrome occurs when excess iron accumulates in tissues and organs to the point of disrupting their normal function. Iron toxicity is a common complication of iron overload syndrome, leading to high mortality rates due to iron buildup in major organs. Beta-thalassemia is an iron overload syndrome that occurs when mutations in the HBB gene cause a reduction or absence of β-hemoglobin (β-hemoglobin). These mutations lead to erythropoiesis and insufficient mature erythrocytes, resulting in ineffective erythropoiesis, which causes anemia and excessive iron uptake leading to iron toxicity. In patients with β-thalassemia, hepcidin is abnormally suppressed relative to the patient's iron load status, resulting in hepcidin deficiency, which in turn allows for excessive iron uptake and the development of systemic iron overload. Other conditions, such as myelodysplastic syndrome (MDS), erythropoietic anemia, and sideroblastic anemia, also exhibit ineffective erythropoiesis through low hepcidin levels leading to iron overload. Hemochromatosis (e.g., type 1 hemochromatosis or hereditary hemochromatosis) is an iron overload disorder characterized by excessive intestinal absorption of dietary iron and a pathological increase in systemic iron storage. Current standards of care for iron overload disorders include transfusions for iron chelation and ineffective erythropoiesis that can further exacerbate iron overload and result in poor patient compliance, and venous stenosis or splenectomy to control symptoms. Treatments currently under development include gene therapy targeting the HBB gene, gene therapy and gene editing targeting other related genes, hepcidin mimics, Fc fusion proteins targeting TGF superfamily ligands to inhibit SMAD signaling, antisense RNA drugs targeting TMPRSS6 (e.g., El-Beshlawy A. et al., Blood Cells, Molecules and Diseases 2019.76:53-58), and iRNA drugs targeting TMPRSS6. Summary of the Invention

[0010] This invention relates to novel antibodies that bind to TMPRSS6 and their antigen-binding fragments, and methods for preparing and using antibodies that bind to TMPRSS6 and their antigen-binding fragments.

[0011] This invention provides anti-TMPRSS6 antibodies, nucleic acids encoding anti-TMPRSS6 antibodies, and methods for preparing and using anti-TMPRSS6 antibodies. As disclosed herein, anti-TMPRSS6 antibodies encompass anti-TMPRSS6 antibodies and fragments thereof capable of binding to TMPRSS6. As disclosed herein, anti-TMPRSS6 antibodies can bind to human TMPRSS6 on the surface of cells expressing human TMPRSS6. This invention provides anti-TMPRSS6 antibodies for therapeutic and diagnostic purposes. As disclosed herein, anti-TMPRSS6 antibodies can be used to treat iron metabolism disorders, such as iron overload disorders, particularly β-thalassemia, including (but not limited to) transfusion-independent thalassemia, and other disorders with ineffective erythropoiesis.

[0012] In one aspect, an anti-TMPRSS6 antibody is provided that can bind to TMPRSS6 on the surface of cells expressing TMPRSS6 and regulate the activity of at least one component involved in iron metabolism, wherein the component may be a molecule or biological process related to the function of TMPRSS6. In some embodiments, the anti-TMPRSS6 antibody disclosed herein can regulate the activity of at least one component involved in regulating hepcidin expression. In some embodiments, the anti-TMPRSS6 antibody disclosed herein can substantially inhibit the repression of hepcidin expression by TMPRSS6. In some embodiments, the anti-TMPRSS6 antibody disclosed herein can increase hepcidin expression. In some embodiments, the anti-TMPRSS6 antibody disclosed herein can increase the activity of the hepcidin promoter. In some embodiments, the anti-TMPRSS6 antibody disclosed herein can substantially inhibit the repression of hepcidin expression induced by the BMP / SMAD pathway by TMPRSS6. The anti-TMPRSS6 antibody disclosed herein can regulate hepcidin expression in a dose-dependent manner, including (but not limited to) substantially inhibiting TMPRSS6-induced hepcidin expression, increasing hepcidin expression, increasing hepcidin promoter activity, or substantially inhibiting TMPRSS6-induced hepcidin expression induced by the BMP / SMAD pathway. In some embodiments, the anti-TMPRSS6 antibody disclosed herein can regulate hepcidin expression in a dose-dependent manner. In some embodiments, the anti-TMPRSS6 antibody disclosed herein can increase serum hepcidin levels in a dose-dependent manner upon administration to an individual. In some embodiments, the anti-TMPRSS6 antibody disclosed herein can decrease serum iron levels in a dose-dependent manner upon administration to an individual. In some embodiments, the anti-TMPRSS6 antibody disclosed herein can increase hepatic hepcidin RNA levels in a dose-dependent manner upon administration to an individual. In some embodiments, the anti-TMPRSS6 antibody disclosed herein can reduce hepatic non-proheme iron, increase serum hepcidin, increase hepatic hepcidin RNA, reduce splenomegaly, increase red blood cell count (RBC), increase hematocrit (HCT), reduce red blood cell distribution width (RDW), and increase the production of mature red blood cells (increased erythropoiesis) when administered to individuals with known or suspected iron overload (especially β-thalassemia).

[0013] On the other hand, the anti-TMPRSS6 antibodies disclosed herein exhibit cross-reactivity with at least one non-human TMPRSS6. In some embodiments, the anti-TMPRSS6 antibodies disclosed herein are capable of binding to at least one non-human TMPRSS6 on the surface of cells expressing at least one non-human TMPRSS6. The anti-TMPRSS6 antibodies disclosed herein may be capable of binding to both human TMPRSS6 and mouse TMPRSS6. The anti-TMPRSS6 antibodies disclosed herein may be capable of binding to both human TMPRSS6 and cynomolgus monkey TMPRSS6. The anti-TMPRSS6 antibodies disclosed herein may be capable of binding to each of human TMPRSS6, mouse TMPRSS6, and cynomolgus monkey TMPRSS6.

[0014] On the other hand, the anti-TMPRSS6 antibody disclosed herein specifically binds to TMPRSS6 (interstitial protease-2). In some embodiments, the anti-TMPRSS6 antibody disclosed herein binds to TMPRSS6 (interstitial protease-2) and does not exhibit detectable binding to interstitial protease homologs. In some embodiments, the anti-TMPRSS6 antibody disclosed herein binds to human TMPRSS6 (interstitial protease-2) and does not exhibit detectable binding to human interstitial protease-1 (ST14). In some embodiments, the anti-TMPRSS6 antibody disclosed herein binds to human TMPRSS6 (interstitial protease-2) and does not exhibit detectable binding to human interstitial protease-3 (TMPRSS7). In some embodiments, the anti-TMPRSS6 antibody disclosed herein binds to human TMPRSS6 (interstitial protease-2) and does not exhibit detectable binding to either human interstitial protease-1 (ST14) or human interstitial protease-3 (TMPRSS7).

[0015] The anti-TMPRSS6 antibodies disclosed herein may be monoclonal antibodies, humanized antibodies, chimeric antibodies, single-chain antibodies, Fab fragments, single-chain variable fragments (scFv), recombinant antibodies, aptamers, single-domain antibodies (VHH, nanobodies), or other TMPRSS6 binding fragments or variants. In some embodiments, the anti-TMPRSS6 antibodies disclosed herein may include a framework in which amino acids have been substituted into an existing antibody framework, particularly a framework affecting properties such as antigen-binding ability. In some embodiments, the anti-TMPRSS6 antibodies disclosed herein may include a complementarity-determining region (CDR) from a source (parental) antibody that has been grafted (fused) into a framework from a different type (class) of antibody and / or an organism different from the parent antibody, particularly a human receptor framework. In some embodiments, the anti-TMPRSS6 antibodies disclosed herein may include a framework in which amino acids have been substituted, mutated, or replaced in regions outside the CDR to affect properties such as antigen binding or antibody structure, for example, in a variable region framework surrounding the CDR and / or in a constant region (especially the Fc region). In some embodiments, one or more of the CDRs have been substituted, mutated, or replaced. In some embodiments, the anti-TMPRSS6 antibody disclosed herein may be a humanized anti-TMPRSS6 antibody variant.

[0016] In some embodiments, the anti-TMPRSS6 antibody disclosed herein comprises at least one polypeptide having an amino acid sequence as set forth in Table 1, Table 2 or Table 3, or a sequence substantially identical (e.g., at least 85%, 90%, 92%, 95%, 97% or 98%, 99%) to the amino acid sequence set forth in Table 1, Table 2 or Table 3. The anti-TMPRSS6 antibody disclosed herein may comprise at least one polypeptide having an amino acid sequence selected from the following, or substantially identical (e.g., at least 85%, 90%, 92%, 95%, 97%, or 98%, 99%) to at least one polypeptide having an amino acid sequence selected from the following: SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:7; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO:12; SEQ ID NO:13; SEQ ID NO:14; SEQ ID NO:16; SEQ ID NO:17; SEQ ID NO:18; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:22; SEQ ID NO:23; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:27; SEQ ID NO:28; SEQ ID NO:29; SEQ ID NO:31; SEQ ID SEQ ID NO:33; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:37; SEQ ID NO:38; SEQ ID NO:39; SEQ ID NO:41; SEQ ID NO:42; SEQ ID NO:43; SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:47; SEQ ID NO:48; SEQ ID SEQ ID NO:51; SEQ ID NO:52; SEQ ID NO:53; SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:57; SEQ ID NO:58; SEQ ID NO:59; SEQ NO:61; SEQ ID NO:63; SEQ ID NO:65; SEQ ID NO:67; SEQ ID NO:69; SEQ ID NO:71; ID NO:73; SEQ ID NO:75; SEQ ID NO:77; SEQ ID NO:79; SEQ ID NO:81; or SEQ ID NO:83.

[0017] In one embodiment, the anti-TMPRSS6 antibody disclosed herein comprises a heavy chain (HC) variable region polypeptide with the amino acid sequence set forth in SEQ ID NO:1 or a sequence substantially identical to that of SEQ ID NO:1, and a light chain (LC) variable region polypeptide with the amino acid sequence set forth in SEQ ID NO:6 or a sequence substantially identical to that of SEQ ID NO:6. In one embodiment, the anti-TMPRSS6 antibody disclosed herein comprises heavy chain complementarity-determining region 1 (HC CDR1) of the amino acid sequence described in SEQ ID NO:2, heavy chain complementarity-determining region 2 (HC CDR2) of the amino acid sequence described in SEQ ID NO:3, and heavy chain complementarity-determining region 3 (HC CDR3) of the amino acid sequence described in SEQ ID NO:4; light chain complementarity-determining region 1 (LC CDR1) of the amino acid sequence described in SEQ ID NO:7, light chain complementarity-determining region 2 (LC CDR2) of the amino acid sequence described in SEQ ID NO:8, and light chain complementarity-determining region 3 (LC CDR3) of the amino acid sequence described in SEQ ID NO:9; or a variant of the antibody comprising 1, 2, 3, 4, 5, or 6 amino acid substitutions in the CDR regions. In a non-limiting embodiment, the anti-TMPRSS6 antibody disclosed herein is the antibody identified herein as MWTx-001, comprising an HC polypeptide having the amino acid sequence described in SEQ ID NO:61 and an LC polypeptide having the amino acid sequence described in SEQ ID NO:63.

[0018] In one embodiment, the anti-TMPRSS6 antibody disclosed herein comprises an HC variable region polypeptide with the amino acid sequence described in SEQ ID NO:11 or a sequence substantially identical to SEQ ID NO:11, and an LC variable region polypeptide with the amino acid sequence described in SEQ ID NO:16 or a sequence substantially identical to SEQ ID NO:16. In one embodiment, the anti-TMPRSS6 antibody disclosed herein comprises HC CDR1 with the amino acid sequence described in SEQ ID NO:12, HC CDR2 with the amino acid sequence described in SEQ ID NO:13, and HC CDR3 with the amino acid sequence described in SEQ ID NO:14; LC CDR1 with the amino acid sequence described in SEQ ID NO:17, LC CDR2 with the amino acid sequence described in SEQ ID NO:18, and LC CDR3 with the amino acid sequence described in SEQ ID NO:19, or a variant of the antibody comprising 1, 2, 3, 4, 5, or 6 amino acid substitutions in the CDR region. In one non-limiting embodiment, the anti-TMPRSS6 antibody disclosed herein has an antibody identified herein as MWTx-002, which comprises an HC polypeptide having the amino acid sequence set forth in SEQ ID NO:65 and an LC polypeptide having the amino acid sequence set forth in SEQ ID NO:67.

[0019] In one embodiment, the anti-TMPRSS6 antibody disclosed herein comprises an HC variable region polypeptide with the amino acid sequence described in SEQ ID NO:21 or a sequence substantially identical to SEQ ID NO:21, and an LC variable region polypeptide with the amino acid sequence described in SEQ ID NO:26 or a sequence substantially identical to SEQ ID NO:26. In one embodiment, the anti-TMPRSS6 antibody disclosed herein comprises HC CDR1 with the amino acid sequence described in SEQ ID NO:22, HC CDR2 with the amino acid sequence described in SEQ ID NO:23, and HC CDR3 with the amino acid sequence described in SEQ ID NO:24; LC CDR1 with the amino acid sequence described in SEQ ID NO:27, LC CDR2 with the amino acid sequence described in SEQ ID NO:28, and LC CDR3 with the amino acid sequence described in SEQ ID NO:29, or a variant of the antibody comprising substitutions of 1, 2, 3, 4, 5, or 6 amino acids in the CDR region. In one non-limiting embodiment, the anti-TMPRSS6 antibody disclosed herein is the antibody identified herein as MWTx-003, which comprises an HC polypeptide having the amino acid sequence set forth in SEQ ID NO:69 and an LC polypeptide having the amino acid sequence set forth in SEQ ID NO:71.

[0020] In one embodiment, the anti-TMPRSS6 antibody disclosed herein comprises an HC variable region polypeptide with the amino acid sequence described in SEQ ID NO:31 or a sequence substantially identical to SEQ ID NO:31, and an LC variable region polypeptide with the amino acid sequence described in SEQ ID NO:36 or a sequence substantially identical to SEQ ID NO:36. In one embodiment, the anti-TMPRSS6 antibody disclosed herein comprises HC CDR1 with the amino acid sequence described in SEQ ID NO:32, HC CDR2 with the amino acid sequence described in SEQ ID NO:33, and HC CDR3 with the amino acid sequence described in SEQ ID NO:34; LC CDR1 with the amino acid sequence described in SEQ ID NO:37, LC CDR2 with the amino acid sequence described in SEQ ID NO:38, and LC CDR3 with the amino acid sequence described in SEQ ID NO:39, or a variant of the antibody comprising 1, 2, 3, 4, 5, or 6 amino acid substitutions in the CDR region. In one non-limiting embodiment, the anti-TMPRSS6 antibody disclosed herein is an antibody identified herein as a humanized anti-TMPRSS6 antibody variant hzMWTx-001Var, comprising an HC polypeptide having the amino acid sequence set forth in SEQ ID NO:73 and an LC polypeptide having the amino acid sequence set forth in SEQ ID NO:75.

[0021] In one embodiment, the anti-TMPRSS6 antibody disclosed herein comprises an HC variable region polypeptide with the amino acid sequence described in SEQ ID NO:41 or a sequence substantially identical to SEQ ID NO:41, and an LC variable region polypeptide with the amino acid sequence described in SEQ ID NO:46 or a sequence substantially identical to SEQ ID NO:46. In one embodiment, the anti-TMPRSS6 antibody disclosed herein comprises HC CDR1 with the amino acid sequence described in SEQ ID NO:42, HC CDR2 with the amino acid sequence described in SEQ ID NO:43, and HC CDR3 with the amino acid sequence described in SEQ ID NO:44; LC CDR1 with the amino acid sequence described in SEQ ID NO:47, LC CDR2 with the amino acid sequence described in SEQ ID NO:48, and LC CDR3 with the amino acid sequence described in SEQ ID NO:49, or a variant of the antibody comprising 1, 2, 3, 4, 5, or 6 amino acid substitutions in the CDR region. In one non-limiting embodiment, the anti-TMPRSS6 antibody disclosed herein is an antibody identified herein as a humanized anti-TMPRSS6 antibody variant hzMWTx-002Var, which comprises an HC polypeptide having the amino acid sequence set forth in SEQ ID NO:77 and an LC polypeptide having the amino acid sequence set forth in SEQ ID NO:79.

[0022] In one embodiment, the anti-TMPRSS6 antibody disclosed herein comprises an HC variable region polypeptide with the amino acid sequence described in SEQ ID NO:51 or a sequence substantially identical to SEQ ID NO:51, and an LC variable region polypeptide with the amino acid sequence described in SEQ ID NO:56 or a sequence substantially identical to SEQ ID NO:56. In one embodiment, the anti-TMPRSS6 antibody disclosed herein comprises HC CDR1 with the amino acid sequence described in SEQ ID NO:52, HC CDR2 with the amino acid sequence described in SEQ ID NO:53, and HC CDR3 with the amino acid sequence described in SEQ ID NO:54; LC CDR1 with the amino acid sequence described in SEQ ID NO:57, LC CDR2 with the amino acid sequence described in SEQ ID NO:58, and LC CDR3 with the amino acid sequence described in SEQ ID NO:59, or a variant of the antibody comprising 1, 2, 3, 4, 5, or 6 amino acid substitutions in the CDR region. In one non-limiting embodiment, the anti-TMPRSS6 antibody disclosed herein is an antibody identified herein as a humanized anti-TMPRSS6 antibody variant hzMWTx-003Var, which comprises an HC polypeptide having the amino acid sequence set forth in SEQ ID NO:81 and an LC polypeptide having the amino acid sequence set forth in SEQ ID NO:83.

[0023] On the other hand, anti-TMPRSS6 antibodies (comprising variants and fragments as disclosed herein) are provided for the treatment of iron metabolism disorders, such as iron overload disorders, particularly β-thalassemia and other conditions of ineffective erythropoiesis. Methods and compositions for using anti-TMPRSS6 antibodies as disclosed herein for therapeutic use are provided, said therapeutic use including (but not limited to) the treatment of iron metabolism disorders, such as iron overload disorders, particularly β-thalassemia and other conditions of ineffective erythropoiesis. In some embodiments, pharmaceutical compositions comprising the anti-TMPRSS6 antibodies disclosed herein and suitable carriers and / or excipients are provided.

[0024] On the other hand, methods for treating iron metabolism disorders are provided, including administering an effective amount of the anti-TMPRSS6 antibody disclosed herein to an individual in need, wherein the effective amount of the anti-TMPRSS6 antibody modulates the activity of components involved in iron metabolism. In some embodiments, the method for treating iron overload disorders includes administering an effective amount of the anti-TMPRSS6 antibody disclosed herein, wherein the effective amount of the anti-TMPRSS6 antibody modulates the activity of components involved in iron metabolism. In some embodiments, the method for treating iron overload disorders includes administering an effective amount of the anti-TMPRSS6 antibody disclosed herein, wherein the effective amount of the anti-TMPRSS6 antibody modulates the activity of at least one component involved in regulating hepcidin expression. In some embodiments, the method includes administering an effective amount of the anti-TMPRSS6 antibody that inhibits the inhibition of hepcidin expression by TMPRSS6. In some embodiments, the effective amount of the anti-TMPRSS6 antibody increases hepcidin expression. In some embodiments, the method includes administering an effective amount of the anti-TMPRSS6 antibody that increases the activity of the hepcidin promoter. In some embodiments, the method includes administering an effective amount of an anti-TMPRSS6 antibody that inhibits the suppression of hepcidin expression induced by the BMP / SMAD pathway. In some embodiments, the method includes administering an effective amount of an anti-TMPRSS6 antibody to an individual, resulting in one or more biological effects associated with iron overload syndrome, including (but not limited to) reduced serum iron, reduced hepatic non-proheme iron, increased serum hepcidin, increased hepatic hepcidin RNA, reduced splenomegaly, increased red blood cell count (RBC), increased hematocrit (HCT), reduced red blood cell distribution width (RDW), and / or increased production of mature red blood cells (increased erythropoiesis).

[0025] On the other hand, methods are provided for treating diseases or disease conditions involving aberrant inhibition of hepcidin expression, such methods comprising administering to an individual in need an effective amount of the anti-TMPRSS6 antibody disclosed herein, wherein administering an effective amount of the anti-TMPRSS6 antibody modulates the activity of at least one component involved in the aberrant inhibition of hepcidin expression and reduces the aberrant inhibition of hepcidin expression. In a particular embodiment, the method induces an increase in hepcidin expression.

[0026] On the other hand, methods are provided for treating iron metabolism disorders associated with suppressed hepcidin levels, such methods comprising administering to an individual in need an effective amount of the anti-TMPRSS6 antibody disclosed herein, wherein the administration of the effective amount of the anti-TMPRSS6 antibody modulates the activity of at least one component involved in suppressing hepcidin levels. In some embodiments, the method includes administering an effective amount of the anti-TMPRSS6 antibody that increases serum hepcidin levels, increases hepatic hepcidin RNA, and decreases serum iron levels.

[0027] On the other hand, methods are provided for treating iron metabolism disorders (including disorders associated with and / or characterized by ineffective erythropoiesis), said disorders including (but not limited to) β-thalassemia. According to this aspect, such methods include administering an effective amount of the anti-TMPRSS6 antibody disclosed herein to an individual known or suspected of having an iron metabolism disorder associated with and / or characterized by ineffective erythropoiesis, wherein the administration results in one or more changes in the individual related to iron metabolism and / or erythropoiesis. In some embodiments, methods are provided in which an effective amount of the anti-TMPRSS6 antibody is administered to treat or improve at least one biological effect or symptom associated with the disorder. In particular embodiments, the practice results in one or more changes, including (but not limited to) a reduction in hepatic non-proheme iron, an increase in serum hepcidin, an increase in hepatic hepcidin RNA, a reduction in splenomegaly, an increase in red blood cell count (RBC), an increase in hematocrit (HCT), a decrease in red blood cell distribution width (RDW), and an increase in the production of mature red blood cells (increased erythropoiesis).

[0028] On the other hand, methods are provided for diagnosing or screening individuals for iron overload syndrome. In some embodiments, the methods include administering an anti-TMPRSS6 antibody to an individual known or suspected of having iron overload syndrome and measuring one or more biological effects or symptoms associated with iron overload syndrome.

[0029] On the other hand, one or more isolated nucleic acid molecules encoding at least a portion of at least one of the anti-TMPRSS6 antibodies disclosed herein are provided. In some embodiments, the isolated nucleic acid molecule encoding at least a portion of at least one of the anti-TMPRSS6 antibodies disclosed herein comprises a nucleotide sequence as set forth in Table 1, Table 2 or Table 3, or a sequence substantially identical (e.g., at least 85%, 90%, 92%, 95%, 97%, or 98%, 99%) to the nucleotide sequences set forth in Table 1, Table 2 or Table 3. In some embodiments, the isolated nucleic acid molecule encoding at least one of the heavy chain (HC) sequences of the anti-TMPRSS6 antibody disclosed herein may include a nucleotide sequence selected from at least one of the following: SEQ ID NO:5 or a sequence substantially identical to SEQ ID NO:5; SEQ ID NO:15 or a sequence substantially identical to SEQ ID NO:15; SEQ ID NO:25 or a sequence substantially identical to SEQ ID NO:25; SEQ ID NO:35 or a sequence substantially identical to SEQ ID NO:35; SEQ ID NO:45 or a sequence substantially identical to SEQ ID NO:45; SEQ ID NO:55 or a sequence substantially identical to SEQ ID NO:55; SEQ ID NO:62 or a sequence substantially identical to SEQ ID NO:62; SEQ ID NO:66 or a sequence substantially identical to SEQ ID NO:66; SEQ ID NO:70 or a sequence substantially identical to SEQ ID NO:70; SEQ ID NO:74 or a sequence substantially identical to SEQ ID NO:74; SEQ ID NO:75 ... NO:78 or a sequence substantially identical to SEQ ID NO:78, or SEQ ID NO:82 or a sequence substantially identical to SEQ ID NO:82.In some embodiments, the isolated nucleic acid molecule encoding at least one of the light chain (LC) sequences of the anti-TMPRSS6 antibody or its antigen-binding fragment disclosed herein may include a nucleotide sequence selected from at least one of the following: SEQ ID NO:10 or a sequence substantially identical to SEQ ID NO:10; SEQ ID NO:20 or a sequence substantially identical to SEQ ID NO:20; or SEQ ID NO:30 or a sequence substantially identical to SEQ ID NO:30; SEQ ID NO:40 or a sequence substantially identical to SEQ ID NO:40; SEQ ID NO:50 or a sequence substantially identical to SEQ ID NO:50; SEQ ID NO:60 or a sequence substantially identical to SEQ ID NO:60; SEQ ID NO:64 or a sequence substantially identical to SEQ ID NO:64; SEQ ID NO:68 or a sequence substantially identical to SEQ ID NO:68; SEQ ID NO:72 or a sequence substantially identical to SEQ ID NO:72; SEQ ID NO:76 or a sequence substantially identical to SEQ ID NO:76; SEQ SEQ ID NO:80 or a sequence substantially identical to SEQ ID NO:80, or SEQ ID NO:84 or a sequence substantially identical to SEQ ID NO:84.

[0030] In another embodiment, a vector comprising one or more nucleic acid molecules is provided, said one or more nucleic acid molecules encoding at least one amino acid sequence of the anti-TMPRSS6 antibody disclosed herein. In some embodiments, a vector comprising one or more nucleic acid molecules is provided, said one or more nucleic acid molecules encoding at least one of the heavy chain (HC) or light chain (LC) sequences of the anti-TMPRSS6 antibody disclosed herein. In some embodiments, a vector comprising nucleic acid molecules is provided, said nucleic acid molecules encoding at least a portion of at least one of the amino acid sequences described in Tables 1, 2, or 3, or at least a portion of an amino acid sequence substantially identical to the amino acid sequences described in Tables 1, 2, or 3. In some embodiments, a vector comprising nucleic acid molecules is provided, said nucleic acid molecules encoding at least a portion of at least one of the HC or LC sequences described in Tables 1, 2, or 3, or at least a portion of an amino acid sequence substantially identical to at least one of the HC or LC sequences described in Tables 1, 2, or 3.

[0031] In another embodiment, at least one host cell is provided containing a vector comprising one or more nucleic acid molecules, said nucleic acid molecules encoding the amino acid sequence of the anti-TMPRSS6 antibody disclosed herein. In some embodiments, a host cell is provided containing a vector comprising nucleic acid molecules encoding at least a portion of at least one of the HC or LC sequences as described in Tables 1, 2, or 3, or at least a portion of an amino acid sequence substantially identical to at least one of the HC or LC sequences as described in Tables 1, 2, or 3. In some embodiments, at least one host cell is capable of supporting vector expression and recombination of an anti-TMPRSS6 antibody or an antigen-binding fragment thereof encoded by the vector. In some embodiments, at least one host cell is capable of supporting vector expression and recombination of an anti-TMPRSS6 antibody or an antigen-binding fragment thereof encoded by a vector comprising nucleic acid molecules, said nucleic acid molecules encoding at least a portion of at least one of the HC or LC sequences as described in Tables 1, 2, or 3, or at least a portion of an amino acid sequence substantially identical to at least one of the HC or LC sequences as described in Tables 1, 2, or 3. In some embodiments, host cells are temporarily transfected via a vector comprising one or more nucleic acid molecules encoding the amino acid sequence of the anti-TMPRSS6 antibody or its antigen-binding fragment disclosed herein, wherein the host cells are capable of supporting vector expression and recombination of the anti-TMPRSS6 antibody or its antigen-binding fragment encoded by the vector. Attached Figure Description

[0032] Figure 1 The results of a cascade screening of anti-TMPRSS6 antibodies are presented, in which in vitro functional assays targeting HAMP promoter activity were used to evaluate antibodies that bind to human TMPRSS6, and antibodies that affect HAMP promoter activity were evaluated for cross-reactivity with non-human TMPRSS6.

[0033] Figures 2A to 2F The effect of anti-TMPRSS6 antibody on HAMP promoter activity was demonstrated by dual-luciferase reporter assays performed in HepG2 cells at a range of antibody concentrations. In each graph, hollow circles represent results using anti-TMPRSS6 antibody, and hollow squares represent results using the same concentration of mouse IgG or human IgG1 as a negative (non-specific binding) control. Figure 2A This study demonstrates the effect of MWTx-001 anti-TMPRSS6 antibody on HAMP promoter activity within a certain antibody concentration range. Figure 2B This study demonstrates the effect of the MWTx-002 anti-TMPRSS6 antibody on HAMP promoter activity within a certain antibody concentration range. Figure 2CThis study demonstrates the effect of MWTx-003 anti-TMPRSS6 antibody on HAMP promoter activity within a certain antibody concentration range. Figure 2D This study demonstrates the effect of hzMWTx-001Var anti-TMPRSS6 antibody on HAMP promoter activity within a certain antibody concentration range. Figure 2E This study demonstrates the effect of hzMWTx-002Var anti-TMPRSS6 antibody on HAMP promoter activity within a certain antibody concentration range. Figure 2F This study demonstrates the effect of hzMWTx-003Var anti-TMPRSS6 antibody on HAMP promoter activity within a certain antibody concentration range.

[0034] Figures 3A to 3M The results of the binding affinity assay for the anti-TMPRSS6 antibody are presented. Figures 3A to 3F The results show the binding affinity of anti-TMPRSS6 antibody against human TMPRSS6 expressed on HEK293T cells, determined using two different methods. In each graph, hollow circles represent results using anti-TMPRSS6 antibody at a specific concentration range, and hollow squares represent results using the same concentration of mouse IgG as a negative control. Figures 3A to 3C Demonstrates the use of cell surface ELISA (measuring HRP-labeled secondary antibodies) to measure MWTx-001 ( Figure 3A ), MWTx-002 ( Figure 3B ) and MWTx-003 ( Figure 3C The results of the binding of ) to human TMPRSS6, including the calculated EC50 for each antibody. 50 The value is used as an assessment of binding affinity. Figures 3D to 3F Demonstrates the use of FACS (a secondary antibody that measures APC binding) to measure MWTx-001 ( Figure 3D ), MWTx-002 ( Figure 3E ) and MWTx-003 ( Figure 3F The results of the binding of ) to human TMPRSS6, including the calculated EC50 for each antibody. 50 The value is used as an assessment of binding affinity. Figure 3G to 3M Demonstration and use The RED96e assay determined the affinity and binding kinetics of the anti-TMPRSS6 antibody against human ecto-TMPRSS6-FLAG at analyte concentrations of 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.13 nM, 1.56 nM, and 0.78 nM. Figure 3G Demonstrates the binding kinetics of the MWTx-001 anti-TMPRSS6 antibody against ecto-TMPRSS6-FLAG. Figure 3HDemonstrates the binding kinetics of the MWTx-002 anti-TMPRSS6 antibody against ecto-TMPRSS6-FLAG. Figure 3I Demonstrates the binding kinetics of the MWTx-003 anti-TMPRSS6 antibody against ecto-TMPRSS6-FLAG. Figure 3J Demonstrates the binding kinetics of hzMWTx-001Var anti-TMPRSS6 antibody against ecto-TMPRSS6-FLAG. Figure 3K Demonstrates the binding kinetics of hzMWTx-002Var anti-TMPRSS6 antibody against ecto-TMPRSS6-FLAG. Figure 3L Demonstrates the binding kinetics of hzMWTx-003Var anti-TMPRSS6 antibody against ecto-TMPRSS6-FLAG. Figure 3M Overview of affinity measurements for all anti-TMPRSS6 antibodies.

[0035] Figures 4A to 4U The results of the cross-reactivity assay for the anti-TMPRSS6 antibody are presented. Figures 4A to 4I This section presents the cross-reactivity assays of anti-TMPRSS6 antibodies MWTx-001, MWTx-002, and MWTx-003 against human and non-human TMPRSS6 expressed on HEK293T cells. Histograms show the FACS results (thin lines and light fill; indicated by antibody name) of the single antibody co-cultured with HEK293T cells expressing the TMPRSS6 target, and the FACS results (thick lines and dark fill; indicated by Ctrl) of the same antibody co-cultured with control HEK293T cells that do not express the TMPRSS6 protein. Figures 4A to 4C Demonstrating the use of HEK293T cells stably expressing human TMPRSS6 (HuTMPRSS6-(His)6) and MWTx-001 ( Figure 4A ), MWTx-002 ( Figure 4B ) and MWTx-003 ( Figure 4C The result of ). Figures 4D to 4F Demonstrating HEK293T cells stably expressing mouse TMPRSS6 (MoTMPRSS6-(His)6) and MWTx-001 ( Figure 4D ), MWTx-002 ( Figure 4E ) and MWTx-003 ( Figure 4F The result of ). Figures 4G to 4I Demonstrating HEK293T cells using transient expression of cynomolgus macaque TMPRSS6 (CynoTMPRSS6-(His)6) and MWTx-001 ( Figure 4G ), MWTx-002 ( Figure 4H) and MWTx-003 ( Figure 4I The result of ). Figures 4J to 4U Demonstrating the effectiveness of anti-TMPRSS6 antibody against non-human (mouse) expression on HEK293T cells. Figure 4J , 4L 4N, 4P, 4R, 4T) or crab-eating macaques ( Figure 4K , 4M The results of the cross-reactivity of TMPRSS6 (4O, 4Q, 4S, 4U) were obtained using a cell surface ELISA (measuring HRP-labeled secondary antibodies) to measure the MWTx-001 anti-TMPRSS6 antibody ( Figures 4J to 4K ), MWTx-002 anti-TMPRSS6 antibody ( Figure 4L to 4M ), MWTx-003 anti-TMPRSS6 antibody ( Figures 4N to 4O ), hzMWTx-001Var anti-TMPRSS6 antibody ( Figures 4P to 4Q ), hzMWTx-002Var anti-TMPRSS6 antibody ( Figure 4R to 4S ) and hzMWTx-003Var anti-TMPRSS6 antibody ( Figure 4T to 4U The binding of anti-TMPRSS6 to non-human TMPRSS6 is shown in the graphs. Hollow circles represent results using anti-TMPRSS6 antibodies, and hollow squares represent results using mouse IgG or human IgG1 as negative (non-specific binding) controls. The calculated EC50 for each antibody is shown in the graphs. 50 The value is used as an assessment of binding affinity.

[0036] Figures 5A to 5R Demonstrating the anti-TMPRSS6 monoclonal antibody MWTx-001 ( Figures 5A to 5C ), MWTx-002 ( Figures 5D to 5F ), MWTx-003 ( Figure 5G to 5I Anti-TMPRSS6 antibody and its humanized variant hzMWTx-001Var ( Figures 5J to 5L ), hzMWTx-002Var Figures 5M to 5O ), hzMWTx-003Var ( Figures 5P to 5R The results of FACS analysis of the binding of anti-TMPRSS6 antibody to HEK293T cells expressing homologous mesenchymal protease. HEK293T cells stably expressing human TMPRSS6 (mesenchymal protease-2) Figure 5A , 5D 5G, 5J, 5M, and 5P were used as positive controls, and interstitial protease (ST14) was overexpressed. Figure 5B , 5E 5H, 5K, 5N, 5Q) and / or interstitial proteinase-3 (TMPRSS7) Figure 5C , 5FHEK293T cells containing the proteins 5I, 5L, 5O, and 5R were used to test their binding to homologous mesenchymal proteases. (See Figures...) Figures 5A to 5R In this study, HEK293T cells that did not express interstitial protease (HEK293T) were used as a negative control, with the results of the control clearly indicated (Ctrl) being shown.

[0037] Figures 6A to 6L This study demonstrates that anti-TMPRSS6 antibody treatment increases hepcidin expression in mice in a dose-dependent manner. Figures 6A to 6C Demonstrating the MWTx-003 anti-TMPRSS6 antibody ( Figures 6A to 6B ) or its humanized variant hzMWTx-003Var anti-TMPRSS6 antibody ( Figure 6C The effect of serum iron. Figure 6D The effect of GFP-TMPRSS6 on serum hepcidin is demonstrated. Figures 6D to 6F Demonstrating the MWTx-003 anti-TMPRSS6 antibody ( Figures 6D to 6E ) or its humanized variant hzMWTx-003Var anti-TMPRSS6 antibody ( Figure 6F The effect of serum iron. Figure 6G The effect of GFP-TMPRSS6 on hepcidin RNA in the liver is demonstrated. Figures 6G to 6I Demonstrating the MWTx-003 anti-TMPRSS6 antibody ( Figures 6G to 6H ) or its humanized variant hzMWTx-003Var anti-TMPRSS6 antibody ( Figure 6I The effect of hepcidin RNA on liver. Figures 6J to 6L Demonstrating the MWTx-003 anti-TMPRSS6 antibody ( Figures 6J to 6K ) or its humanized variant hzMWTx-003Var anti-TMPRSS6 antibody ( Figure 6L Serum concentrations of mouse IgG2b (MoIG2b) Figures 6A to 6B 6D to 6E, 6G to 6H, 6J to 6K) or human IgG1 (HuIGg1) Figure 6C , 6F (6I, 6L) were used as isotype controls, PBS was used as a mediator control, and the GFP vector was used as a vector control. Figure 6A , 6D 6G, 6J).

[0038] Figures 7A to 7R Demonstrating the in vivo efficacy of the anti-TMPRSS6 antibody using a mouse model of β-thalassemia. Figures 7A to 7D Demonstrating the effectiveness of MWTx-003 anti-TMPRSS6 antibody against RBCs using Th3 / + mice. Figure 7A ), HGB Figure 7B HCT Figure 7C) and RDW Figure 7D The impact of ). Figure 7E This study demonstrates the effect of the MWTx-003 anti-TMPRSS6 antibody on spleen weight in Th3 / + mice. Figure 7F This study demonstrates the effect of the MWTx-003 anti-TMPRSS6 antibody on serum iron in Th3 / + mice. Figure 7G This study demonstrates the effect of the MWTx-003 anti-TMPRSS6 antibody on non-proheme iron in the liver using Th3 / + mice. Figure 7H This study demonstrates the effect of the MWTx-003 anti-TMPRSS6 antibody on serum hepcidin in Th3 / + mice. Figure 7I This study demonstrates the effect of the MWTx-003 anti-TMPRSS6 antibody on hepcidin RNA in Th3 / + mice. Figure 7J Serum concentrations of MWTx-003 anti-TMPRSS6 antibody in Th3 / + mice are shown. Figures 7L to 7M The effect of the MWTx-003 anti-TMPRSS6 antibody on erythropoiesis was demonstrated using bone marrow from Th3 / + mice. Figures 70 to 7P The effect of the MWTx-003 anti-TMPRSS6 antibody on erythropoiesis using spleen cells from Th3 / + mice is demonstrated. Figures 7K to 7P The representative curves in the figure show four different cell clusters (I: basophils; II: polychromatic erythrocytes; III: normochromatic erythrocytes and anucleate reticulocytes; and IV: mature erythrocytes) and highlight the corresponding cell number percentages. Wild-type mice were used as positive controls. Figures 7A to 7J , 7K, 7N), and mouse IgG2b (MoIgG2b) was used as an isotype control in treatment ( Figures 7A to 7J 7L, 7O). Figures 7Q to 7R The bar chart shows the bone marrow morphology (BMR) of each treatment regimen (WT, Th3 / +w / MoIgG2b, Th3 / +w / MWTx-003) after 4 weeks. Figure 7Q ) and spleen ( Figure 7R The mean results of cell clusters I, II, III and IV in the study were compared, which allowed for the identification of the transformation of each population. Most notably, the transformation to mature red blood cells (cluster IV) was observed after MWTx-003 treatment.

[0039] Figures 8A to 8D Demonstration and use Results of epitope binning of RED96e, MWTx-001, MWTx-002 and MWTx-003 anti-TMPRSS6 antibodies against human ecto-TMPRSS6-FLAG. Figure 8AThis demonstrates the epitope grouping of the MWTx-001 anti-TMPRSS6 antibody against ecto-TMPRSS6-FLAG. Figure 8B This demonstrates the epitope grouping of the MWTx-002 anti-TMPRSS6 antibody against ecto-TMPRSS6-FLAG. Figure 8C This demonstrates the epitope grouping of the MWTx-003 anti-TMPRSS6 antibody against ecto-TMPRSS6-FLAG. Figure 8D Overview of association signals of anti-TMPRSS6 antibodies MWTx-001, MWTx-002, and MWTx-003. Detailed Implementation

[0040] This invention relates to novel antibodies that bind to TMPRSS6 and their antigen-binding fragments, as well as methods for preparing and using them.

[0041] Terms / Definitions

[0042] Unless otherwise defined, scientific and technical terms used in connection with this invention shall have the meanings commonly understood by one of ordinary skill in the art. Unless the context clearly requires otherwise, the use of singular terms (“a / an” or “the” or other singular terms) shall include multiple references, and plural terms shall include the singular. Thus, for example, reference to “antibody” includes “one or more” antibodies or “a plural” such antibodies. All disclosures mentioned herein are incorporated herein by reference in their entirety.

[0043] Generally, nomenclature and techniques of molecular biology, microbiology, cell and tissue culture, protein and nucleotide chemistry, and recombinant DNA technology available to those skilled in the art can be used for the antibodies, antigen-binding fragments, compositions, and methods disclosed herein. The techniques and procedures described herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references (especially Sambrook et al. (1989), *Molecular Cloning: A Laboratory Manual* (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) and Ausubel et al. (1994), *Current Protocols in Molecular Biology*, Volumes I-III (John Wiley & Sons, NY)). Unless otherwise stated herein, enzymatic reactions and purification techniques are performed according to the manufacturer's instructions or as commonly practiced in the field or as described herein. Techniques and methods for use in pharmaceutical preparations and formulations, as well as for treating individuals, are described herein using conventional nomenclature.

[0044] In the broadest sense, an "antibody" refers to a polypeptide or combination of polypeptides that recognizes and binds to an antigen through one or more immunoglobulin variable regions. These immunoglobulin variable regions can be naturally occurring or non-natural, for example, due to engineering, chimerism, humanization, optimization, CDR grafting, or affinity maturation.

[0045] As disclosed herein, “antibody” can be a whole (intact, full-length) antibody, a single-chain antibody, or an antigen-binding fragment having one or two chains, and can be naturally occurring or non-naturally occurring. An antibody includes at least a sufficient complementarity-determining region (CDR) interspersed with a framework region (FR) for antibody recognition and binding to an antigen. The anti-TMPRSS6 antibody disclosed herein can be (but is not limited to) at least one of the following: monoclonal antibody, polyclonal antibody, humanized antibody, chimeric antibody, single-chain antibody, Fab fragment, single-chain variable fragment (scFv), aptamer, single-domain antibody (VHH or nanobody), recombinant antibody, modified antibody having a peptide / other portion linked to the antibody and / or additional amino acids added to the N-terminus or C-terminus, or other TMPRSS6-binding fragments or variants. The terms whole antibody, full-length antibody, intact antibody, naturally occurring antibody, or equivalent terms should be understood to mean a polypeptide, especially a glycoprotein, comprising at least two heavy chains (HC) and two light chains (LC) linked by disulfide bonds. Each HC comprises a heavy chain variable region (VH) and an HC constant region (CH), and each light chain comprises a light chain variable region (VL) and an LC constant region (CL). The HC and LC variable regions (i.e., VH and VL) contain binding domains that interact with the antigen. The VH and VL regions can be further subdivided into CDR regions characterized by high denaturation, interspersed with generally more conserved FR regions. Each VH and VL typically consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant regions of an antibody mediate the binding of immunoglobulins to host tissues or factors, encompassing various cells of the immune system and the typical complement system. Typically, an antibody comprises at least heavy chain (HC) CDR1, CDR2, and CDR3, and light chain (LC) CDR1, CDR2, and CDR3 sequences, any of which may be naturally occurring or non-naturally occurring. Antibodies may include a small number of CDR sequences, as long as the antibody can recognize and bind to the antigen.

[0046] The anti-TMPRSS6 antibody disclosed herein may be a variant comprising at least one altered CDR or framework sequence, wherein the CDR and / or framework sequence may be optimized by mutating nucleic acid molecules encoding such framework sequences. Variants may be constructed independently from HC and LC moieties derived from dissimilar sources. Techniques for generating variants include (but are not limited to) conserved amino acid substitutions, computer modeling, screening candidate peptides alone or in combination, and codon optimization, and it should be understood that skilled personnel can generate antibody variants as needed. The anti-TMPRSS6 antibody disclosed herein may be a fragment. Antigen-binding function of antibodies can be achieved through fragments such as: Fab fragments; monovalent fragments consisting of VL, VH, CL, and CH1 domains; F(ab)2 fragments; bivalent fragments comprising two Fab fragments linked by disulfide bridges in the hinge region; Fd fragments consisting of VH and CH1 domains; single-chain variable fragments (scFv) consisting of VL and VH domains of a single arm of the antibody; single-domain antibody (dAb) fragments consisting of VH domains; and isolated CDRs (VHH, nanobodies), or aptamers. The antigen-binding portion can be incorporated into single-domain antibodies, maximal antibodies, microbodies, nanobodies, intracellular antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, v-NARs, and bi-scFvs (see, for example, Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136). The antigen-binding portion of an antibody can be grafted onto a peptide-based backbone to form a monofunctional antibody (see, for example, U.S. Patent No. 6,703,199, which describes a fibronectin peptide monofunctional antibody).

[0047] The term antibody encompasses a wide range of polypeptides that can be distinguished biochemically. The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. Those skilled in the art will understand that there are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, several of which can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, each of which is well-characterized and known to confer functional specificity. Modifications of each of these classes and isotypes are readily identifiable and within the scope of this invention. Although all immunoglobulin classes are within the scope of this invention, this invention will primarily target IgG immunoglobulin molecules.

[0048] The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain (HC) and / or light chain (LC) involved in forming the immunoreactive site is derived from a specific source or species, while the remaining portions of the HC and / or LC are derived from different sources or species. In some embodiments, the target binding region or site will be derived from a non-human source (e.g., mouse or non-human primate), and the constant region will be human.

[0049] As used herein, the phrase “humanized antibody” refers to an antibody or antibody variant derived from a non-human antibody (typically a mouse monoclonal antibody), wherein the CDR derived from the parent, non-human antibody is grafted (fused) into a framework that includes a variable region derived from a human immunoglobulin framework (particularly the receptor human framework or human common framework). The techniques and principles used for designing, preparing, and testing humanized antibodies are known (Jones PT, Dear PH, Foote J, Neuberger MS, Winter G. Replacing the complementarity-determining regions in a human antibody with those from a mouse. Nature. May 29–June 4, 1986; 321(6069):522-5; Almagro JC, Fransson J. Humanization of antibodies. Frontiers in Bioscience. January 1, 2008; 13:1619-33). It should be understood that the receptor architecture at multiple sites can be modified to produce humanized antibodies with improved characteristics (e.g., high affinity for the target, low clearance, low toxicity, etc.) for the desired use. The anti-TMPRSS6 antibody disclosed herein is a humanized variant.

[0050] “Affinity” refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, “binding affinity” refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). Affinity can be measured by commonly used methods known in the art, including those described herein. The calculated concentration at approximately 50% of maximum binding (the calculated EC50) is... 50 This can be used to assess affinity. The affinity of molecule X for its partner Y can usually be determined by the dissociation constant (Kd or KD, representing the k-value measured for the interaction). off / kon )express.

[0051] "Individual" refers to a mammal, which includes (but is not limited to) primates (e.g., humans and non-human primates such as monkeys), domesticated animals (e.g., cattle, sheep, cats, dogs, pigs, llamas, and horses), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual is a human. The phrases "to an individual in need," "to a patient in need," "to a patient in need of treatment," or "individual in need of treatment" may include individuals who would benefit from administration of the anti-TMPRSS6 antibody disclosed herein for the treatment of iron overload syndromes. It should be understood that administration of the anti-TMPRSS6 antibody encompasses administration to "individuals in need" (which may be interpreted as referring to individuals known or suspected of having iron overload syndromes, particularly β-thalassemia) based on indicators such as symptoms, family history, or genotype. It should be further understood that the anti-TMPRSS6 antibody may be administered to individuals who are not yet aware of or suspected of having iron metabolism disorders for purposes that may include (but are not limited to) prophylactic or preventative purposes, screening, diagnostic, research purposes, or to achieve results different from those for treating a condition.

[0052] For example, the “effective amount” of an anti-TMPRSS6 antibody in a pharmaceutical formulation refers to the amount required at the necessary dose and for the necessary duration to effectively achieve the desired therapeutic or preventative outcome. It should be understood that “effective amount” refers to the amount of anti-TMPRSS6 antibody or a pharmaceutical composition including an anti-TMPRSS6 antibody that will elicit a biological response or desired therapeutic effect in cells, tissues, systems, non-human animal individuals, non-human mammalian individuals, or the measured human individual. The terms “therapeutic effective amount,” “pharmacologically effective amount,” and “physiologically effective amount” are used interchangeably to refer to the amount of anti-TMPRSS6 antibody required to provide a critical level of the active agent in the bloodstream or in the target tissue. The precise amount will depend on many factors, such as the specific anti-TMPRSS6 antibody (active agent); the composition and physical properties of the composition; the intended population of the individual / patient to be treated; considerations such as disease condition, age, sex, and individual weight, and the like, and can be readily determined by those skilled in the art based on information provided herein or otherwise available in relevant literature. As used in this context, the terms “improvement,” “increase,” or “decrease” indicate a value or parameter relative to a baseline measurement, such as a measurement of the same individual prior to the start of the treatment described herein, or a measurement of a control individual (or multiple control individuals) in the absence of the treatment described herein.

[0053] The terms "pharmaceutical composition" or "pharmaceutical formulation" refer to preparations of such form that allow the biological activity of the active ingredient contained therein to be effective, particularly anti-TMPRSS6 antibodies. It should be understood that a pharmaceutical composition may contain more than one active ingredient, such as more than one anti-TMPRSS6 antibody, or a combination of an anti-TMPRSS6 antibody with another active ingredient acting on a different target. Such combinations may be (but are not limited to) a combination of an anti-TMPRSS6 antibody with another active ingredient having the desired effect on hematopoietic processes (especially erythropoiesis), a combination of an anti-TMPRSS6 antibody with a gene therapy agent (e.g., an agent for gene therapy targeting the HBB gene), or a combination of an anti-TMPRSS6 antibody with an Fc fusion protein targeting TGF superfamily ligands to stimulate erythropoiesis. "Pharmaceutically acceptable carrier" refers to a component in a pharmaceutical formulation that is not toxic to the individual, other than the active ingredient. It should be understood that a pharmaceutically acceptable carrier may be (but is not limited to) a buffer, excipient, stabilizer, adjuvant, or preservative.

[0054] As used herein, the term "treat" or similar terms may refer to a result that is beneficial to a particular individual within the defined group of conditions. Treatment of iron metabolism disorders may not only refer to reducing, alleviating, slowing, interrupting, inhibiting, mitigating, stopping, or reversing any of the progression or severity of existing symptoms, conditions, ailments, or diseases, but may further encompass the prevention or delay of the onset of symptoms of one or more iron overload disorders, and / or the reduction of the severity or frequency of symptoms of one or more iron overload disorders. The terms "treatment" or "method of treatment" or equivalents may encompass one or more uses of the anti-TMPRSS6 antibody disclosed herein, including (but not limited to) therapeutic, preventative, prophylactic, diagnostic, imaging, and screening uses.

[0055] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating the nucleic acid linked to the vector sequence in a host cell upon introduction of the vector. Vectors that guide the expression of the operatively linked nucleic acid are referred to herein as "expression vectors".

[0056] Anti-TMPRSS6 antibody

[0057] This invention provides antibodies and antigen-binding fragments capable of binding to TMPRSS6 on the cell surface and regulating the activity of at least one component involved in iron metabolism (particularly at least one component involved in iron overload syndromes associated with aberrant inhibition of hepcidin expression). Anti-TMPRSS6 antibodies capable of binding to TMPRSS6 on the cell surface and regulating the activity of at least one component involved in regulating hepcidin expression can be used in methods for treating iron overload syndromes associated with aberrant inhibition of hepcidin expression. Anti-TMPRSS6 antibodies capable of binding to TMPRSS6 on the cell surface and regulating the inhibition of hepcidin expression by TMPRSS6 can be used to therapeutically target TMPRSS6 in methods for treating iron overload syndromes associated with aberrant inhibition of hepcidin expression.

[0058] Once an antibody or fragment specific to TMPRSS6 (especially human TMPRSS6 expressed on the cell surface) has been obtained, the desired biological activity that regulates the activity of at least one component involved in its iron metabolism can be tested using several methods known to skilled personnel.

[0059] It should be understood that, as used herein, “modulate / modulating” or similar terms may refer to one or more effects that may occur when the anti-TMPRSS6 antibody disclosed herein binds to its target. Depending on the component under consideration, “modulate” and its equivalents may refer to different modes of action and effects; that is, modulation may refer to neutralizing, reversing, inhibiting, blocking, reducing, antagonizing, or otherwise interfering with the activity of certain components involved in iron metabolism, while for other components involved in iron metabolism, the term modulation may refer to increasing, enhancing, or having a synergistic effect on these components.

[0060] It should be understood that the term "component" can refer not only to the target molecule TMPRSS6, but also to downstream processes or pathways involved in iron metabolism. Therefore, the component within the meaning of process or pathway can be (but is not limited to) regulating hepcidin expression, TMPRSS6's inhibition of hepcidin expression, the process of hepcidin expression, regulating hepcidin content, increasing hepcidin content, the activity of the hepcidin promoter or the inhibition of hepcidin expression induced by the BMP / SMAD pathway by TMPRSS6, regulating the content of non-proheme iron in the liver, participating in one or more processes of splenomegaly, or participating in one or more hematopoietic processes that regulate red blood cell count (RBC), hematocrit (HCT), red blood cell distribution width (RDW), and erythropoiesis (especially the production of mature red blood cells).

[0061] The anti-TMPRSS6 antibody disclosed herein can be used to therapeutically target at least one component involved in iron metabolism, particularly at least one component involved in iron overload syndromes. In some embodiments, the anti-TMPRSS6 antibody disclosed herein can be used to therapeutically target at least one component involved in regulating hepcidin expression and modulate the activity of the component to achieve increased hepcidin expression. In some embodiments, the anti-TMPRSS6 antibody disclosed herein can be used to modulate the activity of the hepcidin promoter to achieve increased hepcidin expression. It should be understood that the anti-TMPRSS6 antibody disclosed herein can be used to therapeutically target TMPRSS6 and thereby regulate the downstream activity of other components of hepcidin expression, including (but not limited to) regulating hepatic non-proheme iron content, participating in one or more processes of splenomegaly, or participating in one or more hematopoietic processes of regulating red blood cell count (RBC), hematocrit (HCT), red blood cell distribution width (RDW), and erythropoiesis (especially the production of mature red blood cells).

[0062] Using anti-TMPRSS6 antibodies as disclosed herein to therapeutically target at least one component involved in iron metabolism allows for precise modulation of the target component. It should be understood that by using anti-TMPRSS6 antibodies as disclosed herein to precisely target TMPRSS6 and its downstream effects on at least one component involved in regulating hepcidin expression, it is possible to avoid unwanted effects, delivery difficulties, and / or efficacy, as well as regulatory barriers associated with other methods currently in use or under development for treating iron overload conditions, such as transfusions that may further exacerbate iron overload, iron chelation therapy with poor patient compliance, invasive venous cut-offs or splenectomies that only control symptoms, gene therapies targeting the HBB gene with potentially permanent pleiotropic effects in multiple systems, gene therapies and gene editing with unknown off-target effects, Fc fusion proteins targeting TGF superfamily ligands to inhibit SMAD signaling that does not reduce the need for iron chelation therapy to control iron overload, and other methods that are difficult to control or deliver, such as hepcidin mimics and antisense or iRNA drugs targeting TMPRSS6. It should be understood that the use of anti-TMPRSS6 antibodies for precise therapeutic targeting does not preclude the possibility of using anti-TMPRSS6 antibodies in methods and compositions used for combination therapy, such as in combination with another active ingredient acting on a different target, in combination with antibodies binding to different targets, in combination with gene therapy agents and methods targeting the HBB gene, or in combination with Fc fusion proteins that target TGF superfamily ligands to stimulate erythrocyte production.

[0063] The anti-TMPRSS6 antibodies disclosed herein allow for the development of individualized treatments (e.g., dosage, frequency of administration) that can be easily continued and discontinued, and combined with other therapies. In some strategic embodiments, the anti-TMPRSS6 antibodies disclosed herein can be combined with other therapies that address multiple therapeutic goals and / or resolve the deficiencies or adverse effects of one of the therapies in a combination therapy.

[0064] Exemplary Examples of Anti-TMPRSS6 Antibodies and Their Uses

[0065] Non-limiting exemplary embodiments of the anti-TMPRSS6 antibody of the present invention are now (specifically) disclosed in the examples, tables and figures.

[0066] Antibodies that can bind to TMPRSS6

[0067] As demonstrated in the examples, the functional cascade can be used to identify and characterize the anti-TMPRSS6 antibodies of the present invention, wherein the first step in the cascade includes screening for antibodies capable of binding to human TMPRSS6 on the surface of cells expressing TMPRSS6 (Example 1, ...). Figure 1 The second step involves identifying antibodies that can bind to human TMPRSS6 on the surface of cells expressing TMPRSS6 and regulate the activity of components involved in iron metabolism, in which case the ability to increase hepcidin (HAMP) promoter activity is tested (Example 2). For example, by... Figure 1 The exemplary embodiments shown in the document demonstrate that the first step identifies 143 antibodies (clones) capable of binding to human TMPRSS6 on the surface of cells expressing TMPRSS6, and the second step identifies ten (10) antibodies (selected from the 143) as “active” antibodies (clones) capable of increasing the activity of the hepcidin (HAMP) promoter.

[0068] In the third step of the functional cascade ( Figure 1 The study tested the cross-reactivity of ten (10) "active" antibodies with non-human TMPRSS6 targets from sources relevant to further research, specifically testing cross-reactivity with mouse TMPRSS6 in preclinical efficacy studies in mouse models and cross-reactivity with cynomolgus monkey TMPRSS6 in toxicity (safety) studies. (The text abruptly ends here, likely due to an incomplete translation or source material.) Figure 1The exemplary embodiments shown in Example 4 and illustrated in Figure 4 demonstrate that three (3) clones (selected from 10) exhibit cross-reactivity with at least one non-human TMPRSS6 and are named MWTx-001, MWTx-002, and MWTx-003. Each of the monoclonal antibodies was sequenced and the CDR (Kabat number) on each HC and LC was identified. The HC and LC sequences were identified as follows: MWTx-001 (SEQ ID NO: 61(HC) and 63(LC)); MWTx-002 (SEQ ID NO: 65(HC) and 67(LC)); and MWTx-003 (SEQ ID NO: 69(HC) and 71(LC)). The fusionoma cell line producing the MWTx-001 monoclonal antibody was deposited on May 27, 2020, under the Budapest Treaty, with ATCC accession number PTA-126759, at the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Virginia, 20110, United States of America. The fusion tumor cell line that produces the MWTx-002 monoclonal antibody was deposited on May 27, 2020, under the Budapest Treaty, with ATCC depositary number PTA-126760 at the United States Culture Collection, 10801 University Avenue, Manassas, Virginia, United States (20110), USA. The fusion tumor cell line that produces the MWTx-003 monoclonal antibody was deposited on May 27, 2020, under the Budapest Treaty, with ATCC depositary number PTA-126761, at the American Culture Collection (UCC) 10801, University Avenue, Manassas, Virginia, United States (20110), USA.

[0069] Humanized variants

[0070] When administered to human individuals, the humanized antibody, including the CDR derived from a non-human source grafted into the human-derived antibody framework, is expected to be non-immunogenic. As demonstrated by the exemplary embodiment disclosed in Example 2, humanized anti-TMPRSS6 antibody variants were successfully generated, tested, optimized, and selected. Multiple candidate HC and LC variants were developed, each having the same CDR sequence but with variable region framework sequences varying at more than 90% of framework locations, and these variants were tested in different HC / LC combinations to identify combinations with the desired characteristics. Following the initial design and testing, variants exhibiting the desired antigen-binding affinity are selected for further evaluation and development, including (but not limited to) modifications to some parental CDR sequences to avoid potentially undesirable events, such as aspartate isomerization, and modifications to some constant regions (Fc) to achieve the desired function, such as minimizing antibody-dependent cell cytotoxicity (ADCC), to achieve the humanized variants hzMWTx-001Var (SEQ ID NO:73(HC) and 75(LC)), hzMWTx-002Var (SEQ ID NO:77(HC) and 79(LC)) and hzMWTx-003Var (SEQ ID NO:81(HC) and 83(LC)).

[0071] Anti-TMPRSS6 antibody that increases hepcidin promoter activity

[0072] As disclosed herein, antibodies for treating iron overload syndrome characterized by reduced hepcidin expression can modulate the activity of at least one component involved in hepcidin expression, wherein the component may be the activity of the hepcidin promoter. As demonstrated by exemplary embodiments using in vitro analyses disclosed in Example 2, anti-TMPRSS6 antibodies MWTx-001, MWTx-002, MWTx-003, hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var increase HAMP promoter activity in a dose-dependent manner. Figures 2A to 2F While the same concentration of the same type of control does not increase HAMP promoter activity, the same concentration of the control does not increase HAMP promoter activity.

[0073] Anti-TMPRSS6 antibodies with high affinity for the target in the relevant biological context

[0074] The anti-TMPRSS6 antibody exhibits high affinity for a biologically appropriate target (i.e., human TMPRSS6 expressed on the cell surface). This can be demonstrated by using Example 3 and... Figure 3MThe exemplary embodiments of measuring affinity using three different methods disclosed herein demonstrate that the monoclonal antibodies MWTx-001, MWTx-002, and MWTx-003, as well as the humanized variants hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var, consistently exhibit favorable affinity properties for therapeutically effective antibodies or antibody fragments.

[0075] Anti-TMPRSS6 antibodies with cross-reactivity with non-human targets

[0076] Therapeutic antibodies or antibody fragments are expected to have sufficient cross-reactivity with non-human targets (non-human homologs) from sources that will be relevant to further research (e.g., preclinical efficacy studies, animal models of disease, toxicology studies, etc.) such that the antibody or antibody fragment should recognize, for example, mouse homologs and / or primate homologs, such as those from cynomolgus monkeys. As demonstrated by the exemplary embodiments disclosed in Example 4, MWTx-001, hzMWTx-001Var, MWTx-003, and hzMWTx-003Var exhibit detectable cross-reactivity with mouse TMPRSS6, while MWTx-001, MWTx-002, MWTx-003, hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var exhibit detectable cross-reactivity with cynomolgus monkey TMPRSS6.

[0077] Anti-TMPRSS6 antibody specifically binds to TMPRSS6 (interstitial proteinase-2).

[0078] Antibodies that exhibit high specificity for binding to target proteins in the same organism and low cross-reactivity with homologous proteins are expected to have reduced or no off-target effects. The anti-TMPRSS6 antibody provided herein demonstrates high specificity for human TMPRSS6 (interstitial proteinase-2), thus making it suitable for use in targeted compositions and methods. As disclosed in Example 5 and Figures 5A to 5R The exemplary embodiments described herein demonstrate that the monoclonal antibodies MWTx-001, MWTx-002, and MWTx-003, and their humanized variants hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var, exhibit specific binding to human TMPRSS6 (interstitial protease-2) and do not exhibit detectable cross-reactivity with homologous human interstitial proteases, i.e., these antibodies do not exhibit detectable binding to interstitial protease-1 (ST14) or interstitial protease-3 (TMPRSS7).

[0079] Anti-TMPRSS6 antibody with in vivo dose-dependent effects on hormones and symptoms associated with iron overload syndrome

[0080] Antibodies that are expected to increase serum hepcidin (a hormone that controls iron absorption and mobilizes iron from iron stores) levels reduce, improve, or prevent symptoms of iron overload, particularly reducing, improving, or preventing symptoms of elevated serum iron levels. As demonstrated by the exemplary embodiment shown in Example 6, administration of the anti-TMPRSS6 monoclonal antibody MWTx-003 or the humanized variant hzMWTx-003Var to wild-type individuals (i.e., individuals not yet known to have or suspected of having iron overload) resulted in an increase in serum hepcidin levels compared to isotype controls. Figures 6A to 6C ), serum iron levels decreased ( Figures 6D to 6F ) and increased levels of hepcidin RNA in the liver ( Figures 6G to 6I These effects are dose-dependent, which can be interpreted as indicating that, without being bound by the mechanism of action, the dose-dependent in vivo action of anti-TMPRSS6 antibodies indicates that a skilled person can determine the effective amount (dose) for a given individual.

[0081] Anti-TMPRSS6 antibody with in vivo efficacy in a β-thalassemia disease model

[0082] Antibodies and antibody fragments that are expected to alleviate symptoms of one or more iron overload syndromes in vivo when administered to individuals exhibiting animal models of the disease (i.e., individuals known or suspected of having iron overload syndrome) are clinically effective for therapeutic use. As demonstrated in Example 7 using a Th3 / + mouse model of β-thalassemia, administration of the anti-TMPRSS6 monoclonal antibody MWTx-003, compared to isotype controls, produced a variety of effects, including (but not limited to) reducing hepatic non-proheme iron, increasing serum hepcidin, increasing hepatic hepcidin RNA, reducing splenomegaly, increasing red blood cell count (RBC), increasing hematocrit (HCT), reducing red blood cell distribution width (RDW), and increasing the production of mature red blood cells (increased erythropoiesis). Each of these effects can be understood as improving the symptoms of the disease. The symptoms of the disease manifest in multiple biological systems, including (but not limited to) effects in the liver (on hepatic non-proheme iron and hepcidin RNA), effects in the blood (on serum iron levels, circulating hormone levels, especially serum hepcidin levels, RBCs, HCTs, and RDWs), effects on spleen size and function (splenomegaly), and erythropoiesis in multiple sites including (but not limited to) the bone marrow and spleen (on the abundance of different precursor cell types and the abundance of mature erythrocytes in erythropoietic sites). Administration of anti-TMPRSS6 antibody improves multiple symptoms in individuals across the entire disease model, causing the measured symptom levels to shift from those seen in isotype controls of the disease model (untreated disease) towards those seen in wild-type littermates, representing normal levels in genetically similar individuals with unknown or suspected disease. Without being bound by theories or mechanisms of action, it should be understood that ineffective erythropoiesis is the driving force behind the abnormal hepcidin inhibition, leading to increased iron uptake and iron overload. Therefore, treatments that improve erythroblast differentiation and maturation into erythrocytes should be therapeutically beneficial for treating iron overload syndromes. This invention discloses, in a non-limiting exemplary embodiment, an anti-TMPRSS6 antibody therapy that increases erythroblast differentiation and maturation into erythrocytes while also reducing iron load.

[0083] Composition

[0084] Compositions are provided comprising a safe and effective amount of the anti-TMPRSS6 antibody of the present invention and a pharmaceutically acceptable carrier or excipient suitable for the intended use of each composition. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, excipients, stabilizers, preservatives, or combinations thereof. It should be understood that the pharmaceutical formulation should be matched to the drug delivery mode.

[0085] The anti-TMPRSS6 antibody disclosed herein can be administered by any suitable means, including (but not limited to) injection or non-enteral infusion. Non-enteral infusion may include intramuscular, intravenous, intra-arterial, intraperitoneal, subcutaneous administration, or non-enteral delivery to the liver. The anti-TMPRSS6 antibody disclosed herein can be formulated for introduction into liver tissue or vascular structures for local delivery to target tissue. The anti-TMPRSS6 antibody disclosed herein can be administered using a device, in reservoir form, or in a sustained-release formulation (e.g., a semi-permeable matrix or microcapsule containing an antibody-containing solid hydrophobic polymer) to allow for slow and / or measurable and / or local delivery. The anti-TMPRSS6 antibody disclosed herein can be formulated and administered using colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsion form.

[0086] method

[0087] Methods for treating iron metabolism disorders using an effective amount of the anti-TMPRSS6 antibody disclosed herein are provided. Without being bound by a specific mechanism of action, the provided methods for targeting TMPRSS6 with the anti-TMPRSS6 antibody disclosed herein have multiple downstream effects, particularly on components (molecules, systems, processes) involved in iron metabolism and erythropoiesis. Without being bound by a specific mechanism of action, methods for treating iron metabolism disorders using an effective amount of the anti-TMPRSS6 antibody disclosed herein to modulate the activity of components involved in iron metabolism are provided. Specifically, methods for treating iron overload disorders associated with excessive iron accumulation in tissues and organs are provided, said iron overload disorders including those associated with or characterized by ineffective erythropoiesis, which may include (but are not limited to) β-thalassemia, particularly transfusion-dependent thalassemia, myelodysplastic syndrome (MDS), dyserythropoiesis, and sideroblastic anemia. Without being limited to a single mechanism of action, a method is provided to treat iron overload conditions associated with low hepcidin levels (especially conditions associated with suppressed hepcidin expression, including diseases or conditions involving abnormal suppression of hepcidin expression) by administering an anti-TMPRSS6 antibody that can increase hepcidin expression.

[0088] Methods for treating iron metabolism disorders, as provided herein, include administering an effective amount of the anti-TMPRSS6 antibody disclosed herein to an individual in need, wherein administering an effective amount of the anti-TMPRSS6 antibody improves at least one biological effect (symptom) associated with the disorder. Methods for treating iron metabolism disorders associated with inhibited hepcidin levels are provided, wherein administering an effective amount of the anti-TMPRSS6 antibody disclosed herein to an individual in need results in at least one of the following: increased hepcidin promoter activity, increased hepcidin transcription, increased hepcidin RNA content, and increased hepcidin levels (especially serum hepcidin levels). Methods for treating individuals with known or suspected iron overload disorders are provided, wherein administering an effective amount of the anti-TMPRSS6 antibody results in one or more biological effects, including (but not limited to) reducing hepatic non-proheme iron, increasing serum hepcidin, increasing hepatic hepcidin RNA, reducing splenomegaly, increasing red blood cell count (RBC), increasing hematocrit (HCT), reducing red blood cell distribution width (RDW), and increasing the production of mature red blood cells (increased erythropoiesis). A method is provided for treating individuals with known or suspected iron overload characterized by ineffective erythropoiesis, wherein administration of an effective amount of anti-TMPRSS6 antibody produces one or more biological effects, including (but not limited to) reducing hepatic non-proheme iron, increasing serum hepcidin, increasing hepatic hepcidin RNA, reducing splenomegaly, increasing red blood cell count (RBC), increasing hematocrit (HCT), reducing red blood cell distribution width (RDW), and increasing the production of mature red blood cells (increased erythropoiesis).

[0089] Methods and compositions for treating iron metabolism disorders, particularly iron overload disorders, and more specifically iron overload disorders characterized by ineffective erythropoiesis, are provided, wherein administration of an effective amount of anti-TMPRSS6 antibody results in the treatment or relief of more than one disease-related biological effect or symptom. Without wishing to be bound by theories or mechanisms of action, it should be understood that ineffective erythropoiesis, characterized by precursor cell apoptosis, which causes the production of very few mature erythrocytes in the bone marrow, is the driving force behind the abnormal hepcidin inhibition leading to increased iron uptake and iron overload. Based on this understanding, treatments that improve erythroblast differentiation and maturation into erythrocytes should be therapeutically beneficial for treating iron overload disorders. The effectiveness of anti-TMPRSS6 antibody therapy in increasing erythroblast differentiation and maturation into erythrocytes, reducing iron load, and increasing hepcidin expression maximizes the therapeutic benefit of the methods and compositions using the anti-TMPRSS6 antibody disclosed herein.

[0090] The following examples are provided to illustrate, rather than to limit, the claimed invention.

[0091] Example

[0092] Example 1: Antibody production and identification of antibodies binding to TMPRSS6

[0093] The novel monoclonal antibody against TMPRSS6 was generated under contract by the LakePharma Discovery Immunology group (LakePharma, Inc., San Carlos, CA) using in vivo rodent immunization and fusion tumor technology. DNA-based immunization was performed in B6;SJL mice (The Jackson Laboratories) via fluid-dynamic gene transfer via tail vein injection, using a mixture of pLEV113_huTMPRSS6 and pLEV113_moTMPRSS6-TCE plasmid DNA (cloned at LakePharma). Sufficient plasma titers, as determined by fluorescence activated cell sorting (FACS), were obtained to trigger downstream antibody recovery and screening activities. Electrofusion was performed using a NEPA GENE ECFG21 super-electro-cell fusion generator (Nepa Gene Co., Ltd., Ichikawa-City, Chiba, Japan) with aggregated spleen cells from two immunized mice and myeloma fusion partners. Fusion material was seeded in a total of ten (10) 384-well plates in hypoxanthine-aminopterin-thymidine medium specifically selected for fusion tumors within unfused myeloma partner cells. The fusion tumor supernatant was first screened for HuTMPRSS6 reactivity measured by FACS to detect supernatants that produced a positive staining signal on HEK293T cells expressing TMPRSS6 (the plasmid encoding huTMPRSS6-(His)6 (SEQ ID NO:97) transfected into HEK293T cells, selecting HEK293T cells expressing TMPRSS6) and a negative staining signal on the parent (HEK293T) on day 10 post-fusion. Supernatants from fusion tumors that produced positive staining signals on HEK293 cells expressing TMPRSS6 and negative staining on parental cells were designated as “hit” for further screening. 192 hits were identified in the primary FACS screening, and 143 hits were confirmed in the secondary and tertiary FACS screenings.

[0094] Example 2. Functional screening of anti-TMPRSS6 antibodies; identification, production, and sequencing of monoclonal anti-TMPRSS6 antibodies and humanized variants.

[0095] HAMP-luciferase reporter analysis

[0096] Hepcidin promoter-luciferase reporter assays were used to measure the response of the HAMP promoter to various anti-TMPRSS6 antibodies (Du, X. et al., 2008. Science 320:1088-1092; modified to use the human HAMP promoter instead of the mouse HAMP promoter as originally reported). For the HAMP-luciferase reporter assay, a 2.5 kb HAMP promoter fragment (reference genome GRCh38) was spliced ​​upstream of the sequence encoding firefly luciferase. A control construct encoding Renilla luciferase, driven by the thymidine kinase promoter (Promega, E6931), was used as an internal control. These constructs, along with the construct encoding TMPRSS6, were co-transfected into HepG2 cells (ATCC, HB-8065). HepG2 cells transfected with TMPRSS6 were pretreated for approximately 3 hours with purified mAbs diluted at various concentrations in starvation medium containing minimum essential medium (MEM, ATCC) + 1% heat-inactivated fetal bovine serum (FBS, Gibco) + 1mM sodium pyruvate + non-essential amino acid solution (Gibco) + 10mM HEPES (Gibco) + 1% Pen / Strep (Gibco). This was followed by treatment with recombinant hBMP6 (R&D Systems) at a final concentration of 25-60 ng / mL to trigger BMP-SMAD-mediated signaling. Purified mouse IgG (Sigma-Aldrich) or human IgG1 (BioXcell) served as controls. After overnight hBMP6 treatment, cells were lysed and luciferase receptors were added. Luminescence readings from firefly luciferase and Renilla luciferase were recorded separately by measuring total luminescence. The activity was calculated as the ratio of firefly luciferase luminescence to Renida luciferase luminescence (control). The results of these analyses are presented in… Figures 2A to 2F middle.

[0097] In vitro functional screening

[0098] To screen for functionally active fusion tumors, the HAMP-luciferase reporter assay described above was used to test all 143 HuTMPRSS6-binding fusion tumors (“hit”). Ten (10) of the 143 HuTMPRSS6-binding fusion tumors showed increased HAMP promoter activity in their supernatants (data not shown) and were identified as “active clones” for further testing. The cross-reactivity of these ten (10) active clones against the mouse target MoTMPRSS6 described in Example 4 below was tested, and three (3) showed binding to both HuTMPRSS6 and MoTMPRSS6, as measured by FACS. These three cross-reactive clones were further seeded at a density of 1 cell / well in 192 wells of a 384-well disc to generate monoclonal fusion tumor clones. The resulting subclones were identified as MWTx-001, MWTx-002, and MWTx-003, which exhibited the desired functional activity and cross-reactivity against non-human targets (e.g., murine TMPRSS6 (moTMPRSS6) and / or cynomammal TMPRSS6 (cynoTMPRSS6)).

[0099] Sequences of anti-TMPRSS6 antibodies MWTx-001, MWTx-002, and MWTx-003

[0100] The sequences of MWTx-001, MWTx-002, and MWTx-003 were determined as follows: mRNA was isolated from each fusion tumor sample, and reverse transcription polymerase chain reaction (RT-PCR) was performed using a unique set of mouse IgG-specific primers to amplify the target variable region for sequencing. Unique heavy and light chains were identified against each anti-TMPRSS6 antibody. The nucleotide sequences of each heavy and light chain were determined. The amino acid sequences encoded by the nucleotide sequences were determined, and the CDR region was identified using the Kabat numbering system. Table 1 presents the amino acid sequences of the variable regions of the heavy and light chains for each of MWTx-001, MWTx-002, and MWTx-003, as well as the amino acid sequences of the identified CDRs (based on Kabat numbering) and the nucleotide sequences of the variable regions of the heavy and light chains.

[0101]

[0102]

[0103] Generation and screening of humanized anti-TMPRSS6 antibody variants

[0104] Humanization of parental antibodies was achieved by grafting CDRs onto human antibody frameworks. First, 3D homology modeling of the parental antibody structure was performed to establish its structural model. The amino acid sequence of the variable fragment framework was identified based on overall sequence consistency, matching VH-VL interface positions, typical CDR positions of similar classifications, and removal of potential N-glycosylation sites. Humanized antibodies were designed by creating multiple hybrid sequences that fuse selected portions of the parental antibody sequence with the human framework sequence. The isotypes for forming the humanized antibody were selected as IgG1 for the heavy chain and IgG1κ for the light chain. Using a 3D model, both visual and computer modeling were employed to systematically analyze these humanized sequences to isolate the sequences most likely to maintain antigen binding. The goal was to maximize the amount of human sequence in the final humanized antibody while maintaining the initial antibody specificity. Paired humanized VH and VL variants were then expressed and purified for affinity analysis.

[0105] In one round of design, generation, and testing of variants as part of affinity analysis, four VH variants were generated using the VH-CDR of the parental antibody MWTX-003 at corresponding positions in four different human IgG1-derived frameworks (SEQ ID NO: 89-92), and four VL (VK) variants were generated using the VL-CDR of the parental antibody MWTX-003 at corresponding positions in four different human IgG1κ-derived frameworks (SEQ ID NO: 93-96). A total of sixteen (16) humanized variants representing each combination of VH and VL (VK) variants were prepared according to a 4VH×4VK matrix, and antigen-binding properties (k) were evaluated. on k off KD) and found to have KD values ​​in the nanomolar range (4.16E-07 to 1.09E-08).

[0106] Variants exhibiting the desired antigen-binding affinity are selected for further evaluation and development. In some cases, the parental CDR sequence is modified to avoid potentially undesirable events, such as aspartic acid isomerization.

[0107] To silence antibody effector function, specifically antibody-dependent cytotoxicity (ADCC), for all humanized antibody variants, key amino acid residues in the Fc region are identified and mutated (substituted). Guidelines available in the literature regarding Fc mutations aimed at eliminating ADCC can be used to understand the mutations of this invention, such as removing the native Fc N-linked glycosylation site in hIgG1 (N297A mutation), or replacing leucine residues at positions 234 and 235 in the lower hinge region of Fc (LALA double mutation), as in (Tamm A, Schmidt RE. IgG binding sites on human Fc gamma receptors. International Review of Immunology (Int RevImmunol.) 1997; 16(1-2):57-85. doi: 10.3109 / 08830189709045703; Jefferis R, Lund J. Interaction sites on human IgG-Fc for FcgammaR: current model). models). Immunology Letters, June 3, 2002; 82(1-2):57-65. doi: 10.1016 / s0165-2478(02)00019-6). In a variant of the present invention, the N297A mutation is introduced into the Fc of the hzMWTx-001Var and hzMWTx-002Var antibodies, and the LALA mutation is introduced into the Fc of the hzMWTx-003Var antibody to achieve the same goal of reducing or silencing ADCC (Table 3, SEQ ID NO: 73, 77, 81).

[0108] Following evaluation, humanized anti-TMPRSS6 antibody variants hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var were selected for further testing. The sequences and characteristics of the humanized variants are shown in Tables 2 and 3 below.

[0109] Recombination of humanized anti-TMPRSS6 antibody variants

[0110] The expression construct for the humanized anti-TMPRSS6 antibody variant was engineered to have an internal ribosome entry site (IRES) between the LC- and HC-coding DNA sequences. Codon optimization was performed using Geneart DNA synthesis, and the construct was cloned into the pcDNA3.4 mammalian expression vector (Thermo Fisher Scientific). The DNA insert sequence was validated by sequencing. For recombinant antibody production, the expression construct was used for transient transfection using the ExpiCHO expression system (Thermo Fisher Scientific) according to the manufacturer's instructions. The expressed antibody was purified by protein A affinity chromatography. The yield of antibody generated by transient transfection ranged from 50 mg to 300 mg / L, with a purity >95% and an endotoxin content <1 EU / ml.

[0111] Sequences of humanized anti-TMPRSS6 antibody variants hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var

[0112] Humanized anti-TMPRSS6 antibody variants hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var were selected for further testing. The sequences of the variable regions for each variable region are shown in Table 2 below, where identified CDRs are indicated by underlining, and the changes in the CDR sequence of the humanized variants relative to the parent antibody are indicated and discussed.

[0113]

[0114]

[0115]

[0116] Table 3 shows the complete heavy and light chain protein and nucleotide sequences of the anti-TMPRSS6 monoclonal antibodies MWTx-001, MWTx-002, and MWTx-003, as well as the humanized anti-TMPRSS6 antibody variants hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var. The heavy chain protein sequences of the humanized anti-TMPRSS6 antibody variants hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var are shown at the locations of mutations (variations) introduced to reduce ADCC as described above.

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124] Dose-dependent effect of anti-TMPRSS6 antibody on HAMP promoter activity

[0125] Figures 2A to 2F The results are presented using the HAMP-luciferase reporter assay described above to test MWTx-001, MWTx-002, MWTx-003, and their humanized variants hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var at specified concentrations. MWTx-001 ( Figure 2A ), MWTx-002 ( Figure 2B ), MWTx-003 ( Figure 2C ) and humanized variant hzMWTx-001Var ( Figure 2D ), hzMWTx-002Var Figure 2E ), hzMWTx-003Var ( Figure 2F Each of these components increases HAMP promoter activity in a dose-dependent manner. MWTx-001's EC... 50 The calculated value is 3 μg / ml. Figure 2A EC of MWTx-002 50 The calculated value is 1 μg / ml. Figure 2B EC of MWTx-003 50 The calculated value is 2 μg / ml. Figure 2C EC of hzMWTx-001Var 50 The calculated value is 0.8 μg / ml. Figure 2D EC of hzMWTx-002Var 50 The calculated value is 0.3 μg / ml. Figure 2E EC of hzMWTx-003Var 50 The calculated value is 0.3 μg / ml. Figure 2F ).

[0126] Example 3. Binding affinity of anti-TMPRSS6 antibody

[0127] The binding affinity of various anti-TMPRSS6 antibodies to human TMPRSS6 expressed on HEK293T cells was measured using three different methods: cell surface ELISA (…). Figures 3A to 3C ), FACS Figures 3D to 3F ) and biolayer interferometry ( Figure 3G to 3M ).

[0128] Measurement of anti-TMPRSS6 mAb binding affinity using cell surface ELISA

[0129] HEK293T cells stably expressing human TMPRSS6 (manufactured by Lake Pharmaceuticals, as described above; SEQ ID NO: 97) were fixed with 4% paraformaldehyde (PFA) and washed with dPBS (Dulbecco's phosphate-buffered saline, Corning Cellgro) and then cultured with various concentrations of anti-TMPRSS6 antibody diluted in BSA medium (DMEM + 1% Pen / Strep + 10mM HEPES + 1mg / ml BSA (Sigma-Aldrich)). Purified mouse IgG was used as a control (Sigma-Aldrich). After culture, the cells were washed with BSA medium and then cultured with goat anti-mouse IgG bound to HRP as a 2° antibody (Invitrogen) Finally, the cells were washed with dPBS to remove unbound antibodies and developed with ELISA liquid substrate (Sigma-Aldrich), and the reaction was stopped by adding the same volume of 1M H2SO4 to the ELISA liquid substrate. The bound antibodies were detected by OD... 450nm Absorbance measurements were taken at [location]. The results of these analyses are presented in [the table / document / etc.]. Figures 3A to 3C middle.

[0130] Measurement of binding affinity for anti-TMPRSS6 mAb using FACS

[0131] HEK293T cells stably expressing human TMPRSS6 were collected and blocked with dPBS + 3% BSA, followed by incubation with various concentrations of anti-TMPRSS6 antibody diluted in dPBS + 3% BSA. Purified mouse IgG was used as a control. After incubation, cells were washed with dPBS and then incubated with goat anti-mouse IgG (Jackson Immuno Research Inc.) as a 2° antibody bound to APCs. Finally, cells were washed with dPBS to remove unbound antibodies, resuspended in dPBS + 1 mM EDTA, and then used... Flow cytometry (ACEA Biosciences, Inc., San Diego, CA) was used for FACS analysis. Antibody binding was determined by measuring the average APC intensity after excitation at 640 nm and the luminescence (fluorescence) at 675 nm. The results of these analyses are presented in… Figures 3D to 3F middle.

[0132] Affinity and binding kinetics of anti-TMPRSS6 antibody measured using biotomography

[0133] Biological layer interferometry is used for the measurement of affinity of anti-TMPRSS6 antibodies and its applications. Binding kinetics assay using the RED96e system (Sartorius AG, Germany). Prehydrated anti-mouse IgG Fc capture (AMC) biosensor (targeting MWTx-001, MWTx-002, and MWTx-003 anti-TMPRSS6 antibodies). Figures 3G to 3I ) or anti-human IgG Fc capture (AHC) biosensors (targeting hzMWTx-001Var, hzMWTx-002Var and hzMWTx-003Var anti-TMPRSS6 antibodies, Figures 3J to 3L First, equilibrate for 120 seconds in 1×KB (kinetic buffer, 1×PBS pH 7.4 + 0.02% Tween-20 + 0.1% BSA) as the first baseline, then add 10 mg / mL anti-TMPRSS6 antibody (MWTx-001). Figure 3G MWTx-002 Figure 3H MWTx-003 Figure 3I hzMWTx-001Var Figure 3J hzMWTx-002Var Figure 3K hzMWTx-003Var Figure 3L The analyte was loaded onto an AMC or AHC biosensor and held for 240 seconds. Then, a second baseline signal was established for 120 seconds before association with various concentrations of human ecto-TMPRSS6-FLAG (SEQ ID NO: 102) (internally generated by fusing the extracellular domain of human TMPRSS6 with a C-terminal FLAG tag) for 240 seconds. Finally, the analyte was dissociated in 1×KB for 360 seconds. Data analysis was performed using octet data analysis software HT. KD, k on k off and R 2 Overview Figure 3M middle.

[0134] Example 4: Cross-reactivity: Anti-TMPRSS6 antibodies binding to human TMPRSS6 and non-human TMPRSS6

[0135] Cross-reactivity was determined by FACS.

[0136] The selected anti-TMPRSS6 antibody was tested to determine whether any antibody could bind to TMPRSS6 from mice and / or cynomolgus monkeys. HEK293T cells stably expressing human TMPRSS6 (HuTMPRSS6-(His)6) (produced by Lake Pharmaceuticals as described above), HEK293T cells stably expressing mouse TMPRSS6 (MoTMPRSS6-(His)6) (SEQ ID NO:98) (produced by Lake Pharmaceuticals as described above), and HEK293T cells transiently expressing cynomolgus monkey TMPRSS6 (CynoTMPRSS6-(His)6) (SEQ ID NO:99) (internal generation) were collected. HEK293T cells stably expressing human TMPRSS6 served as a positive control, and HEK293T cells served as a negative control (as described above). Cells were blocked with dPBS + 3% BSA and subsequently cultured with anti-TMPRSS6 antibody diluted in dPBS + 3% BSA. After culturing, the cells were washed with dPBS and then recultured with goat anti-mouse IgG (as a 2° antibody) bound to Alexa Fluor-488. Finally, the cells were washed with dPBS to remove unbound antibodies, resuspended in dPBS + 1 mM EDTA, and then used... Flow cytometry (Eisen Biosciences, San Diego, California) was used for FACS analysis. The binding antibody system was determined by excitation at 488 nm and emission measurement at 530 nm (FITC-A). The results of these analyses are presented in… Figures 4A to 4I In the histogram, MWTx-001 was observed. Figure 4D ) and MWTx-003 ( Figure 4F Cross-reactivity between ) and mouse TMPRSS6, however, MWTx-002 ( Figure 4E No detectable cross-reactivity was shown with mouse TMPRSS6. MWTx-001 was observed to exhibit cross-reactivity. Figure 4G ), MWTx-002 ( Figure 4H ) and MWTx-003 ( Figure 4I Cross-reactivity with cynomolgus monkey TMPRSS6.

[0137] Cross-reactivity was determined by cell surface ELISA.

[0138] HEK293T cells stably expressing mouse TMPRSS6 (produced by Lake Pharmaceuticals as described above) were used. Figure 4J , 4L 4N, 4P, 4R, 4T) or HEK293T cells of cynomolgus macaque TMPRSS6 (as described above, generated internally), Figure 4K , 4M 4O, 4Q, 4S, and 4U were fixed with methanol (100%) and washed with dPBS (Durbe Kojic's phosphate-buffered saline, Corning Cellgro), followed by incubation with various concentrations of anti-TMPRSS6 antibody and its humanized variants diluted in BSA medium (DMEM + 1% Pen / Strep + 10mM HEPES + 1mg / ml BSA (Sigma-Aldrich)). Purified mouse IgG ( Figures 4J to 4O ) or human IgG1 ( Figures 4P to 4U ) was used as a control. After culture, the cells were washed with BSA medium and then conjugated with goat anti-mouse (Yingjie, ) as a 2° antibody. Figures 4J to 4O ) or anti-human (Millipore, Figures 4P to 4U IgG was cultured together. Finally, the cells were washed with dPBS to remove unbound antibodies and developed with ELISA liquid substrate (Sigma-Aldrich), then the reaction was stopped by adding an equal volume of 1M H2SO4 to the ELISA liquid substrate. The bound antibodies were detected by incubation at OD... 450nm Absorbance measurements were taken at [location]. The results of these analyses are presented in [the table / document / etc.]. Figures 4J to 4U In the middle. MWTx-001 was observed ( Figure 4J ) and MWTx-003 ( Figure 4N Anti-TMPRSS6 antibody and its humanized variant hzMWTx-001Var ( Figure 4P ) and hzMWTx-003Var ( Figure 4T The cross-reactivity of anti-TMPRSS6 antibody with mouse TMPRSS6, however, MWTx-002 ( Figure 4L Anti-TMPRSS6 antibody or its humanized variant hzMWTx-002Var ( Figure 4R The anti-TMPRSS6 antibody did not show detectable cross-reactivity with mouse TMPRSS6. MWTx-001 ( Figure 4K ), MWTx-002 ( Figure 4M ) and MWTx-003 ( Figure 4O Anti-TMPRSS6 antibody and its humanized variant hzMWTx-001Var ( Figure 4Q ), hzMWTx-002Var Figure 4S) and hzMWTx-003Var ( Figure 4U Cross-reactivity between anti-TMPRSS6 antibody and cynomolgus monkey TMPRSS6.

[0139] Example 5: Target specificity: Anti-TMPRSS6 antibody that binds to homologous interstitial proteases.

[0140] To determine whether the anti-TMPRSS6 antibody binds to the homologous interstitial protease, HEK293T cells (internally generated) that overexpressed interstitial protease (ST14) (SEQ ID NO:100) were collected. Figure 5B , 5E HEK293T cells (5H, 5K, 5N, 5Q) and overexpressing interstitial proteinase-3 (TMPRSS7) (SEQ ID NO:101) Figure 5C , 5F 5I, 5L, 5O, 5R). HEK293T cells stably expressing human TMPRSS6 (interstitial protease-2) (SEQ ID NO:97) (produced by Lake Pharmaceuticals as described above, Figure 5A , 5D 5G, 5J, 5M, and 5P were used as positive controls, and HEK293T cells ( Figures 5A to 5R ) were used as negative controls (as described above). Cells were blocked and infiltrated with dPBS + 3% BSA + 0.1% Tween-20, and then cultured with various anti-TMPRSS6 antibodies diluted in dPBS + 3% BSA + 0.1% Tween-20. Cells were cultured with anti-TMPRSS6 antibodies and their humanized variants at a concentration of approximately 1 μg / ml for 1 hour. After culture, cells were washed with dPBS and with Alexa Fluor-488 (Elite). Figures 5A to 5I Goat anti-mouse IgG bound to or with allophycocyanin (APC) (Jackson Immuno Research), Figures 5J to 5R The cells were cultured together with goat anti-human IgG as a 2° antibody. Finally, the cells were washed with dPBS and resuspended in dPBS + 1 mM EDTA, and then used... Flow cytometry was used for FACS analysis. The bound antibody was detected by excitation at 488 nm and luminescence (FITC-A) was measured at 530 nm. Figures 5A to 5I Or by excitation at 640 nm and measurement of emission at 675 nm (APC-A) Figures 5J to 5R The results of these analyses are presented in [the table / document / etc.]. Figures 5A to 5RIn the histogram, all antibodies showed binding to human TMPRSS6 (interstitial proteinase-2). Figure 5A , 5D 5G, 5J, 5M, 5P) and none of the antibodies showed homologous interstitial protease ST14 ( Figure 5B , 5E 5H, 5K, 5N, 5Q) or TMPRSS7 ( Figure 5C , 5F The binding of 5I, 5L, 5O, and 5R. MWTx-001 anti-TMPRSS6 antibody and its humanized variant hzMWTx-001Var anti-TMPRSS6 antibody demonstrate binding to human TMPRSS6. Figure 5A , 5J And it did not show any interaction with interstitial protease (ST14) ( Figure 5B , 5K ) or interstitial proteinase-3 (TMPRSS7) ( Figure 5C , 5L The binding of MWTx-002 anti-TMPRSS6 antibody and its humanized variant hzMWTx-002Var anti-TMPRSS6 antibody to human TMPRSS6 (interstitial proteinase-2) is demonstrated. Figure 5D , 5M And it did not show any interaction with interstitial protease (ST14) ( Figure 5E , 5N ) or interstitial proteinase-3 (TMPRSS7) ( Figure 5F , 5O The binding of MWTx-003 anti-TMPRSS6 antibody and its humanized variant hzMWTx-003Var anti-TMPRSS6 antibody to human TMPRSS6 (interstitial proteinase-2) is demonstrated. Figure 5G , 5P And it did not show any interaction with interstitial protease (ST14) ( Figure 5H , 5Q ) or interstitial proteinase-3 (TMPRSS7) ( Figure 5I , 5R The combination of ).

[0141] Example 6. Treatment with anti-TMPRSS6 antibody in a mouse pharmacodynamic model

[0142] To study the in vivo pharmacokinetic response of anti-TMPRSS6 antibody, 2 to 10 mg / kg of MWTx-003 anti-TMPRSS6 antibody ( Figures 6A to 6B 6D to 6E, 6G to 6H, 6J to 6K) or their humanized variant hzMWTx-003Var anti-TMPRSS6 antibody ( Figure 6C , 6FIgG2b (6I, 6L) was injected intraperitoneally into wild-type C57BL / 6J mice. Figures 6A to 6B 6D to 6E, 6G to 6H, 6J to 6K) or human IgG1 (Bicosser, Figure 6C , 6F 6I and 6L were used as isotype controls. Twenty hours post-injection, 50 μg of GFP-TMPRSS6 plasmid DNA (internally generated by inserting human TMPRSS6 into the GFP vector) was delivered to mice via hydrodynamic tail vein injection. Forty-four hours post-injection, mice were euthanized, and liver tissue and blood were collected. Liver RNA was purified using EZgene Total RNA Purification Plus from Biomiga (San Diego, California) according to the manufacturer's instructions. Mouse serum was obtained by centrifuging whole blood at 1500 × g for 10 min.

[0143] Effect of anti-TMPRSS6 antibody treatment on serum iron

[0144] Serum iron was measured using an internally developed colorimetric analysis method. Figures 6A to 6C In simple terms, mouse serum or iron standards (31-500 μg / dL) were vortexed for 30 seconds with a mixed acid solution (0.6 M trichloroacetic acid, 0.4 M sodium thioglycolate, 1 M HCl). The mixture was incubated at 37 °C for 10 min, followed by centrifugation at 10,000 × g for 10 min, and then developed in a color solution (1.5 M sodium acetate, 0.5 mM red phenanthrene disulfonate). The color was then measured at OD... 535nm Read the absorbance at the specified location. Calculate serum iron concentration based on the linear iron standard curve. 10 mg / kg MWTx-003 anti-TMPRSS6 antibody ( Figures 6A to 6B ) and its humanized variant hzMWTx-003Var anti-TMPRSS6 antibody ( Figure 6C Treatment significantly reduced serum iron.

[0145] Effect of anti-TMPRSS6 antibody treatment on serum hepcidin

[0146] Serum hepcidin was measured according to the manufacturer's instructions using the Hepcidin-Mouse Competitive ELISA Kit purchased from Intrinsic Lifesciences (La Jolla, CA). Figures 6D to 6FIn simple terms, diluted mouse serum or hepcidin standards are mixed with hepcidin-biotin conjugates and then added to culture dishes coated with anti-mouse hepcidin antibodies. Serum hepcidin or hepcidin standards competitively bind to the coated anti-hepcidin antibodies against the hepcidin-biotin conjugates. The hepcidin-biotin conjugates are detected using streptavidin-bound horseradish peroxidase (HRP) and colorimetrically with TMB, followed by stop solution. Then, at OD... 450nm Absorbance was read at the specified location. Data were analyzed using a Graphpad Prism 8 with four-parameter logic (4-PL) curve fitting, and serum hepcidin concentration was interpolated. Hydrodynamic delivery of GFP-TMPRSS6 significantly reduced serum hepcidin levels. Figure 6D However, using 10 mg / kg MWTx-003 anti-TMPRSS6 antibody ( Figures 6D to 6E ) and its humanized variant hzMWTx-003Var anti-TMPRSS6 antibody ( Figure 6F Treatment reversed hepcidin inhibition and significantly increased serum hepcidin levels.

[0147] Effect of anti-TMPRSS6 antibody treatment on hepatic hepcidin RNA

[0148] Quantitative analysis of hepatic hepcidin RNA by real-time qPCR Figures 6G to 6I In short, cDNA was first synthesized from liver RNA using iScript reverse transcription supermix (Bio-Rad) according to the manufacturer's instructions. Hepcidin transcripts were amplified using the specific primers listed in Table 4 below, and on a Bio-Rad CFX96 qPCR instrument using SsoAdvanced according to the manufacturer's instructions. TM Universal Bio-Rad assay was performed using green supermix. Samples were analyzed in triplicate, and results were normalized to β-actin RNA content (measured by transcription, amplification using primers listed in Table 4, and quantification as described above). Fluid-dynamic delivery of GFP-TMPRSS6 significantly reduced hepatic hepcidin RNA (…). Figure 6G ). 10 mg / kg MWTx-003 anti-TMPRSS6 antibody ( Figures 6G to 6H ) and its humanized variant hzMWTx-003Var anti-TMPRSS6 antibody ( Figure 6ITreatment with Hamp reversed the inhibition and significantly increased the hepatic hepcidin RNA content. The following primers were used for RNA quantification by real-time qPCR: Hepcidin forward primer: 5'-AAG CAG GGC AGA CAT TGC GAT-3' (SEQ ID NO:85); Hepcidin reverse primer: 5'-CAG GAT GTG GCT CTA GGC TAT-3' (SEQ ID NO:86); β-actin forward primer: 5'-ACC CAC ACT GTG CCC ATC TA-3' (SEQ ID NO:87); β-actin reverse primer: 5'-CAC GCT CGG TCA GGA TCT TC-3' (SEQ ID NO:88).

[0149] Serum concentrations of MWTx-003 anti-TMPRSS6 antibody or its humanized variant hzMWTx-003Var anti-TMPRSS6 antibody were quantified using an internally developed cell surface ELISA (as described above). Figures 6J to 6L In short, diluted mouse serum or anti-TMPRSS6 antibody standards were cultured together with 100% methanol-fixed HEK293T cells stably expressing human TMPRSS6 (HEK293T cells were used as a background control). The binding of MWTx-003 anti-TMPRSS6 antibody was detected using HRP-bound goat anti-mouse IgG, and the binding of hzMWTx-003Var anti-TMPRSS6 antibody was detected using HRP-bound goat anti-human IgG. Colorimetry was performed with TMB, followed by stop solution. This was then observed at OD... 450nm Absorbance was read at the specified location. Samples were analyzed in triplicate, and results were normalized to a HEK293T control. Data were analyzed using a Grafpard prism 8 with four-parameter logic (4-PL) curve fitting, and serum anti-TMPRSS6 antibody concentrations were interpolated.

[0150] Example 7. In vivo efficacy of anti-TMPRSS6 antibody using a mouse model of β-thalassemia.

[0151] To investigate the in vivo efficacy of the anti-TMPRSS6 antibody, a β-thalassemia mouse model (B6.129P2-Hbb-b1) was selected. tm1Unc Hbb-b2 tm1Unc / J, JAX Stock No: 002683, The Jackson Laboratories, Bar Harbor, Maine (referred to as Th3 / + mice in this study). Four to five-week-old Th3 / + mice and their wild-type (WT) littermates were fed an adequate iron diet (Teklad TD. 80394). Th3 / + mice were treated every three days with 10 mg / kg MWTx-003 anti-TMPRSS6 antibody or mouse IgG2b isotype control for four weeks, while WT littermates were not treated. At the end of the treatment, the mice were euthanized, and spleen, liver, femur, and blood samples were collected. Total RNA from the liver was purified, and serum was collected as described above.

[0152] Effects on blood count, splenomegaly, serum iron, serum hepcidin, and hepatic hepcidin RNA

[0153] Complete blood cell count (CBC) was performed using the VETSCAN HM5 automated hematology analyzer. Figures 7A to 7D Treatment with MWTx-003 anti-TMPRSS6 antibody significantly increased red blood cell count (RBC). Figure 7A ) and hematocrit (HCT, Figure 7C It also reduces red blood cell distribution width (RDW). Figure 7D However, for heme (HGB) in Th3 / + mice, Figure 7B No significant impact.

[0154] Spleen weight was measured, and treatment with MWTx-003 anti-TMPRSS6 antibody significantly reduced splenomegaly in Th3 / + mice. Figure 7E ).

[0155] Serum iron was measured as described above. Treatment with MWTx-003 anti-TMPRSS6 antibody significantly reduced serum iron ( Figure 7F Using a similar colorimetric analysis to measure non-proheme iron in the liver ( Figure 7G In short, minced small liver tissue was dried overnight at 65°C, followed by digestion at 65°C for 20 hours with a mixed acid solution (3M HCl, 10% trichloroacetic acid). The digestion supernatant was then collected for color development in a colored solution (1.5M sodium acetate, 0.5mM red phenanthrene-2,5-disulfonate). This was then analyzed at OD0.05. 535nm Absorbance was read at [location]. Treatment with MWTx-003 anti-TMPRSS6 antibody significantly reduced non-proheme iron in the liver (…). Figure 7G ).

[0156] Serum hepcidin was measured using the hepcidin-mouse competitive ELISA kit described above. Treatment with MWTx-003 anti-TMPRSS6 antibody significantly increased serum hepcidin ( Figure 7H ).

[0157] Hepatic hepcidin RNA was quantified using real-time qPCR as described above. Treatment with MWTx-003 anti-TMPRSS6 antibody significantly increased hepatic hepcidin RNA. Figure 7I ).

[0158] The serum concentration of MWTx-003 anti-TMPRSS6 antibody was quantified using an internally developed cell surface ELISA as described above. Figure 7J ).

[0159] Effects on erythropoiesis

[0160] To investigate the effect of the MWTx-003 anti-TMPRSS6 antibody on erythropoiesis in Th3 / + mice, bone marrow was collected from the femur (see [link]). Figures 7K to 7M And spleen cells are collected from the spleen (see...) Figures 7N to 7P The collected cells were blocked with rat anti-mouse CD16 / CD32 (BD Biosciences) for 15 min, followed by staining on ice for 30 min with rat anti-mouse TER119 bound to FITC (BD Biosciences) and rat anti-mouse CD44 bound to APC (Yingjie). After washing, the cells were stained on ice for 10 min with the viability marker 7-AAD (BD Biosciences), and then analyzed. Flow cytometry was used for FACS analysis. Ter119 was selected. + 7-ADD - Cells were analyzed, and a density map (FSC-H) relative to cell size was plotted using anti-mouse CD44. The plots were analyzed to identify cell types (cell clusters) and to determine the abundance of each type (cluster). Figures 7K to 7P The representative curves shown correspond to the continuous stages of erythrocyte differentiation, distinguishing four different cell clusters from top to bottom: basophilic erythroblasts (cluster I), polychromatic erythroblasts (cluster II), normochromatic erythroblasts and anucleate reticulocytes (cluster III), and mature erythroblasts (cluster IV). The percentage (%) value of each cluster in the sample is calculated as a measure of the abundance of the cell type in that cluster, such as... Figures 7K to 7PAs shown in the figure. For each sample (bone marrow, spleen) of each animal in each treatment process, the percentage values ​​of each cell cluster (I), (II), (III), (IV) were calculated as follows: WT (untreated) N=9; Th3 / + mice with disease model treated with IgG2b isotype control (Th3+w / MoIgG2b), N=5; Th3 / + mice with disease model treated with MWTx-003 anti-TMPRSS6 antibody (Th3+w / MWTx-003), N=7, and then the average value was calculated. On average, after four weeks, in bone marrow cells, the basophil (I) population showed 7.58% (Th3+ w / MoIgG2b) to 6.52% (Th3+ w / MWTx-003) translocation (7.96% for WT), and the polychromatic (II) population showed 54.20% (Th3+ w / MoIgG2b) to 40.01% (Th3+ w / MWTx-003) translocation. (28.53% for WT), normochromic erythroblasts and anucleated reticulocytes (III) showed translocation of 24.06% (Th3+ w / MoIgG2b) to 29.73% (Th3+ w / MWTx-003) (26.67% for WT) and mature erythroblasts (IV) showed translocation of 4.54% (Th3+ w / MoIgG2b) to 16.44% (27.66% for WT). On average, after four weeks, in the spleen, the basophilic erythroblast (I) population showed 0.71% (Th3+ w / MoIgG2b) to 0.91% (Th3+ w / MWTx-003) translocation (0.46% for WT), and the polychromatic erythroblast (II) population showed 45.76% (Th3+ w / MoIgG2b) to 19.25% (Th3+ w / MWTx-003) translocation (12% for WT). 23%), normochromatic erythroblasts and anucleated reticulocytes (III) showed translocation of 31.16% (Th3+ w / MoIgG2b) to 28.72% (Th3+ w / MWTx-003) (8.67% for WT), and mature erythrocytes (IV) showed translocation of 14.13% (Th3+ w / MoIgG2b) to 44.38% (Th3+ w / MWTx-003) (72.17% for WT). These results for bone marrow are presented in Figure 7Q The bar chart shows these results specifically for the spleen. Figure 7R The bar chart in the image.

[0161] In Th3 / + mice, treatment with the MWTx-003 anti-TMPRSS6 antibody improved ineffective erythropoiesis, with a significant proportion of erythroblasts differentiating and maturing into erythrocytes.

[0162] Example 8. Grouping of anti-TMPRSS6 antibody epitopes

[0163] RED96e for MWTx-001 ( Figure 8A ), MWTx-002 ( Figure 8B ) and MWTx-003 ( Figure 8C Epitope grouping of anti-TMPRSS6 antibody. First, ecto-TMPRSS6-FLAG (as described above) was biotinylated using a biotinylated labeling kit (Abcam). A pre-hydrated streptavidin (SA) biosensor was equilibrated in 1×KB (as described above) for 60 seconds as a first baseline, followed by loading 10 mg / mL of biotinylated ecto-TMPRSS6-FLAG onto the SA biosensor for 300 seconds. Next, a second baseline signal was established for 60 seconds, followed by loading 50 mg / mL of antibody (MWTx-001) into 1×KB. Figure 8A MWTx-002 Figure 8B MWTx-003 Figure 8C Saturation was achieved for 600 seconds. Finally, a third baseline signal was established for 60 seconds, followed by competition with 50 μg / mL MWTx-001, MWTx-002, or MWTx-003 in 1×KB for 300 seconds. The MWTx-001 anti-TMPRSS6 antibody binding to ecto-TMPRSS6-FLAG did not compete with the MWTx-002 or MWTx-003 anti-TMPRSS6 antibodies. Figure 8A The MWTx-002 anti-TMPRSS6 antibody, which binds to ecto-TMPRSS6-FLAG, did not compete with the MWTx-001 anti-TMPRSS6 antibody, but did compete with the MWTx-003 anti-TMPRSS6 antibody. Figure 8B The MWTx-003 anti-TMPRSS6 antibody, which binds to ecto-TMPRSS6-FLAG, did not compete with the MWTx-001 anti-TMPRSS6 antibody, but did compete with the MWTx-002 anti-TMPRSS6 antibody. Figure 8C Data analysis was performed using the HT software for octet data analysis. An overview of association signals is provided below. Figure 8D middle. sequence list <110> Mabwell Therapeutics, Inc. (USA) <120> Anti-TMPRSS6 antibodies and their uses <130> 1121-101PCT <140> <141> <150> 63 / 158,265 <151> 2021-03-08 <150> 63 / 006,695 <151> 2020-04-07 <160> 102 <170> PatentIn version 3.5 <210> 1 <211> 118 <212> PRT <213> Mus musculus <400> 1 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Ala Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Ile Thr Trp Val Lys Gln Arg Pro Gly Gln Asp Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Gly Ser Gly Ser Thr Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Arg Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Ala Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Pro Tyr Asp Ser Asp Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Watch Val Thr Val Watch Watch 115 <210> 2 <211> 8 <212> PRT <213> House mouse (Mus musculus) <400> 2 Gly Tyr Thr Phe Thr Ser Tyr Trp 1 5 <210> 3 <211> 8 <212> PRT <213> House mouse (Mus musculus) <400> 3 Ile Tyr Pro Gly Ser Gly Ser Thr 1 5 <210> 4 <211> 11 <212> PRT <213> House mouse (Mus musculus) <400> 4 Ala Pro Tyr Asp Ser Asp Tyr Ala Met Asp Tyr 1 5 10 <210> 5 <211> 354 <212> DNA <213> House mouse (Mus musculus) <400> 5 caggtccaac tgcagcagcc tggggctgag cttgcgaagc ctggggcttc agtgaagatg 60 tcctgcaagg cttctggcta caccttcacc agctactgga taacctgggt gaagcagagg 120 cctggacaag accttgagtg gattggaaat atttatcctg gtagtggtag tacttactac 180 aatgagaagt tcaagagcaa ggccacactg actgtagaca catcctccag aacagcctac 240 atgcagctca gcagtctgac atctgcggac tctgcggtct attactgtgc cccctatgat 300 tccgactatg ctatggacta ctggggtcaa ggaacctcag tcaccgtctc ctca 354 <210> 6 <211> 107 <212> PRT <213> Mus musculus <400> 6 Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Asn Tyr 20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile 35 40 45 Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Val Ser Gly 50 55 60 Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Tyr 65 70 75 80 Glu Asp Val Gly Ile Tyr Phe Cys Leu Gln Tyr Asp Glu Phe Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 7 <211> 6 <212> PRT <213> House mouse (Mus musculus) <400> 7 Gln Asp Ile Asn Asn Tyr 1 5 <210> 8 <211> 3 <212> PRT <213> House mouse (Mus musculus) <400> 8 Arg Ala Asn 1 <210> 9 <211> 9 <212> PRT <213> House mouse (Mus musculus) <400> 9 Leu Gln Tyr Asp Glu Phe Pro Leu Thr 1 5 <210> 10 <211> 321 <212> DNA <213> House mouse (Mus musculus) <400> 10 gacatcaaga tgacccagtc tccatcttcc atgtatgcat ctctaggaga gagagtcact 60 atcacttgca aggcgagtca ggacattaat aactatttaa gctggttcca gcagaaacca 120 gggaaatctc ctaagaccct gatctatcgt gcaaacagat tggtagatgg ggtcccatca 180 agggtcagtg gcagtggatc tgggcaagat tattctctca ccatcagcag cctggagtat 240 gaagatgtgg gaatttattt ttgtctacag tatgatgagt ttcctctcac gttcggtgct 300 gggaccaagc tggagctgaa a 321 <210> 11 <211> 121 <212> PRT <213> Mus musculus <400> 11 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Ile His Trp Val Lys Glu Arg Thr Glu Gln Gly Leu Glu Trp Phe 35 40 45 Gly Arg Ile Asp Pro Glu Asp Gly Glu Ser Glu Tyr Ala Pro Lys Phe 50 55 60 Gln Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Asp Ser Met Met Val Thr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Leu Thr Val Ser Ser Glu 115 120 <210> 12 <211> 8 <212> PRT <213> House mouse (Mus musculus) <400> 12 Gly Phe Asn Ile Lys Asp Tyr Tyr 1 5 <210> 13 <211> 8 <212> PRT <213> House mouse (Mus musculus) <400> 13 Ile Asp Pro Glu Asp Gly Glu Ser 1 5 <210> 14 <211> 13 <212> PRT <213> House mouse (Mus musculus) <400> 14 Thr Arg Gly Asp Ser Met Met Val Thr Tyr Phe Asp Tyr 1 5 10 <210> 15 <211> 360 <212> DNA <213> House mouse (Mus musculus) <400> 15 gaggttcagc tgcagcagtc tggggcagaa cttgtgaagc caggggcctc agtcaagttg 60 tcctgcacag cctctggctt caacattaaa gactactata tacactgggt gaaagagagg 120 actgaacagg gcctggagtg gtttggaagg attgatcctg aggatggtga aagtgaatat 180 gccccgaaat tccagggcaa ggccacttta acagcagaca catcctccaa tacagcctac 240 ctgcagctca gcagcctgac atctgaggac actgccgtct attactgtac tagaggagac 300 tctatgatgg ttacctactt tgactactgg ggccaaggca ccactctcac ggtctcctca 360 <210> 16 <211> 107 <212> PRT <213> Mus musculus <400> 16 Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Phe Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Ser 50 55 60 Thr Gly Ser Gly Thr Asp Tyr Ala Leu Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Arg Ser Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 17 <211> 6 <212> PRT <213> Mus musculus <400> 17 Gln Asp Val Ser Thr Ala 1 5 <210> 18 <211> 3 <212> PRT <213> Mus musculus <400> 18 Trp Ala Phe 1 <210> 19 <211> 9 <212> PRT <213> Mus musculus <400> 19 Gln Gln His Tyr Arg Ser Pro Trp Thr 1 5 <210> 20 <211> 321 <212> DNA <213> Mus musculus <400> 20 gacattgtga tgacccagtc tcacaaattc atgtccacat cagtaggaga cagggtcagc 60 atcacctgca aggccagtca ggatgtgagt actgctgtag cctggtatca acaaaaacca 120 gggcaatctc ctaaactact gatttactgg gctttcaccc gtcacactgg agtccctgat 180 cgcttcacaa gcactggatc tgggacagat tatgctctca ccatcagcag tgtgcaggct 240 gaagacctgg cactttatta ctgtcagcaa cattatcgca gtccgtggac gttcggtgga 300 ggcaccaaac tggaaatcaa a 321 <210> 21 <211> 118 <212> PRT <213> Mus musculus <400> 21 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Glu Asp Tyr 20 25 30 Tyr Ile His Trp Val Lys Glu Arg Thr Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Glu Asp Gly Glu Thr Thr Tyr Ala Pro Gln Phe 50 55 60 Gln Gly Lys Ala Thr Ile Ile Pro Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ala Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ile Tyr Leu Asp Pro Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <210> 22 <211> 8 <212> PRT <213> Mus musculus <400> 22 Gly Phe Asn Ile Glu Asp Tyr Tyr 1 5 <210> 23 <211> 8 <212> PRT <213> Mus musculus <400> 23 Ile Asp Pro Glu Asp Gly Glu Thr 1 5 <210> 24 <211> 11 <212> PRT <213> Mus musculus <400> 24 Ala Arg Ser Ile Tyr Leu Asp Pro Met Asp Tyr 1 5 10 <210> 25 <211> 354 <212> DNA <213> Mus musculus <400> 25 gaagttcagc tgcagcagtc tggggcagaa cttgtgaagc caggggcctc agtcaagttg 60 tcctgcacag cttctggctt caacattgaa gactactata tacactgggt gaaggagagg 120 actgaacagg gcctggagtg gattggaagg attgatcctg aggatggtga aactacatat 180 gccccgcagt tccagggcaa ggccactata ataccagaca catcctccaa cacagcctac 240 atgcagctca gcagcctgac atctgaggac gctgccgtct attactgtgc tagatcgatc 300 taccttgatc ctatggacta ctggggtcaa ggaacctcag tcaccgtctc ctca 354 <210> 26 <211> 107 <212> PRT <213> Mus musculus <400> 26 Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Thr Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ile Leu Ile 35 40 45 Tyr Trp Ala Thr Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Ile Ser Gly Thr Thr Tyr Ile Leu Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 27 <211> 6 <212> PRT <213> Mus musculus <400> 27 Gln Asp Val Thr Thr Ala 1 5 <210> 28 <211> 3 <212> PRT <213> Mus musculus <400> 28 Trp Ala Thr 1 <210> 29 <211> 9 <212> PRT <213> Mus musculus <400> 29 Gln Gln His Tyr Ser Thr Pro Tyr Thr 1 5 <210> 30 <211> 321 <212> DNA <213> Mus musculus <400> 30 gacattgtga tgacccagtc tcacaaattc atgtccacat cagtaggaga cagggtcagc 60 atcacctgca aggccagtca ggatgtgact actgctgtcg cctggtatca acaaaaacca 120 ggacagtctc ctaaaatact gatttactgg gcaaccaccc ggcacactgg agtccctgat 180 cgcttcacag gcagtatatc tgggacaact tatattctca ccatcagtag tgtgcaggct 240 gaagacctgg cactttatta ctgtcagcaa cattatagca ctccgtacac gttcggaggg 300 gggaccaagc tggaaataaa a 321 <210> 31 <211> 118 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" "Polypeptide" <400> 31 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Gly Ser Gly Ser Thr Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Ser Lys Ala Thr Ile Thr Arg Asp Thr Ser Ser Arg Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Pro Tyr Asp Ala Asp Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 32 <211> 8 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" peptide <400> 32 Gly Tyr Thr Phe Thr Ser Tyr Trp 1 5 <210> 33 <211> 8 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" peptide <400> 33 Ile Tyr Pro Gly Ser Gly Ser Thr 1 5 <210> 34 <211> 11 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" peptide <400> 34 Ala Pro Tyr Asp Ala Asp Tyr Ala Met Asp Tyr 1 5 10 <210> 35 <211> 354 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" "Polynucleotides" <400> 35 gaagtgcagc tggtgcaatc tggcgccgaa gtgaagaaac ctggcgcctc tgtgaaggtg 60 tcctgcaagg cttccggcta cacctttacc agctactgga tcacctgggt ccgacaggct 120 cctggccaga gactggaatg gatcggcaac atctaccctg gctccggctc cacctactac 180 aacgagaagt tcaagtccaa ggccacaatc acccgggaca cctcttccag aaccgcctac 240 atggaactgt ccagcctgag atctgaggac accgccgtgt actactgcgc cccttacgac 300 gccgactacg ccatggatta ttggggccag ggcaccctgg tcaccgtgtc ctct 354 <210> 36 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of artificial sequence: Synthetic polypeptide" <400> 36 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 Lys Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Arg Ala Asn Arg Leu Val Glu Gly Val Pro Ser Arg 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 Phe Cys Leu Gln Tyr Asp Glu Phe Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 37 <211> 6 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" peptide <400> 37 Gln Asp Ile Ser Asn Tyr 1 5 <210> 38 <211> 3 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" peptide <400> 38 Arg Ala Asn 1 <210> 39 <211> 9 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" peptide <400> 39 Leu Gln Tyr Asp Glu Phe Pro Leu Thr 1 5 <210> 40 <211> 321 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" "Polynucleotides" <400> 40 gacatccaga tgacccagtc tccatcctct ctgtccgcct ctgtgggcga cagagtgacc 60 atcacatgca aggccagcca ggacatctcc aactacctgt cctggttcca gcagaagcct 120 ggcaaggctc ccaagctgct gatctacaga gccaacagac tggtggaagg cgtgccctcc 180 agattctccg gatctggctc tggcaccgac tttaccctga caatctccag cctgcagcct 240 gaggacttcg ctacctactt ctgcctgcaa tacgacgagt tccctctgac ctttggcgga 300 ggcaccaagg tggaaatcaa g 321 <210> 41 <211> 120 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" "Polypeptide" <400> 41 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Glu Asp Ala Glu Ser Glu Tyr Ala Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Asp Ser Met Met Val Thr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 42 <211> 8 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" peptide <400> 42 Gly Phe Asn Ile Lys Asp Tyr Tyr 1 5 <210> 43 <211> 8 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" peptide <400> 43 Ile Asp Pro Glu Asp Ala Glu Ser 1 5 <210> 44 <211> 13 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" peptide <400> 44 Thr Arg Gly Asp Ser Met Met Val Thr Tyr Phe Asp Tyr 1 5 10 <210> 45 <211> 360 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" "Polynucleotides" <400> 45 gaagtgcagc tggtgcaatc tggcgccgaa gtgaagaaac ctggcgcctc tgtgaaggtg 60 tcctgcaagg cctctggctt caacatcaag gactactaca tccactgggt ccgacaggct 120 accggacagg gacttgagtg gatgggcaga atcgaccctg aggacgccga gtctgagtac 180 gcccctaagt ttcagggcag agtgaccatc accgccgaca cctctaccga caccgcctac 240 atggaactgt ccagcctgag atctgaggac accgccgtgt actactgcac cagaggcgac 300 tccatgatgg ttacctactt cgactactgg ggccagggca ccctggtcac agtttcttcc 360 <210> 46 <211> 107 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" "Polypeptide" <400> 46 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 Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Phe Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ala Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Arg Ser Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 47 <211> 6 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" peptide <400> 47 Gln Asp Val Ser Thr Ala 1 5 <210> 48 <211> 3 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" peptide <400> 48 Trp Ala Phe 1 <210> 49 <211> 9 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" peptide <400> 49 Gln Gln His Tyr Arg Ser Pro Trp Thr 1 5 <210> 50 <211> 321 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" "Polynucleotides" <400> 50 gacatccaga tgacccagtc tccatcctct ctgtccgcct ctgtgggcga cagagtgacc 60 gacatccaga tgacccagtc tccatcctct ctgtccgcct ctgtgggcga cagagtgacc 60 atcacatgca aggcctctca ggacgtgtcc accgccgttg cttggtatca gcagaagcct 120 atcacatgca aggcctctca ggacgtgtcc accgccgttg cttggtatca gcagaagcct 120 ggcaaggccc ctaagctgct gatctactgg gccttcacca gacacaccgg cgtgccctct 180 ggcaaggccc ctaagctgct gatctactgg gccttcacca gacacaccgg cgtgccctct 180 aggttctccg gctctggctc tggcaccgat tacgctctga caatctccag cctgcagcct 240 aggttctccg gctctggctc tggcaccgat tacgctctga caatctccag cctgcagcct 240 gaggacttcg ccacctacta ctgccagcag cactacagaa gcccctggac atttggcgga 300 gaggacttcg ccacctacta ctgccagcag cactacagaa gcccctggac atttggcgga 300 ggcaccaagg tggaaatcaa g 321 ggcaccaagg tggaaatcaa g 321 <210> 51<210> 51 <211> 118<211> 118 <212> PRT<212> PRT <213> 人工序列<213> Artificial sequence <220><220> <221> source<221> source <223> / 注释=“人工序列的描述:合成 <223> / Note=“Description of artificial sequence: synthetic 多肽” peptide” <400> 51 <400> 51 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Glu Asp Tyr Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Glu Asp Tyr 20 25 30 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 35 40 45 Gly Arg Ile Asp Pro Glu Asp Ala Glu Thr Thr Tyr Ser Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Ile Pro Asp Thr Ser Ala Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ile Tyr Leu Asp Pro Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 52 <211> 8 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" peptide <400> 52 Gly Phe Asn Ile Glu Asp Tyr Tyr 1 5 <210> 53 <211> 8 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" peptide <400> 53 Ile Asp Pro Glu Asp Ala Glu Thr 1 5 <210> 54 <211> 11 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" peptide <400> 54 Ala Arg Ser Ile Tyr Leu Asp Pro Met Asp Tyr 1 5 10 <210> 55 <211> 354 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" "Polynucleotides" <400> 55 caggtgcagc tggtgcagtc tggcgccgaa gtgaaaaagc ctggcgcctc tgtgaaggtg 60 tcctgcaagg cctctggctt caacatcgag gactactaca tgcactgggt ccgacaggcc 120 cctggccaga gattggaatg gatgggcaga atcgaccccg aggacgccga gacaacctac 180 tctcctaagt tccagggccg cgtgacaatc atccctgaca cctctgccaa caccgcctac 240 atggaactgt ccagcctgag atctgaggac accgccgtgt actactgcgc ccggtctatc 300 tacctggacc ctatggacta ttggggccag ggcaccctgg tcacagtgtc ctct 354 <210> 56 <211> 107 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" "Polypeptide" <400> 56 Asp Ile Gln Met Thr Gln Ser Pro Lys Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Thr Thr Ala 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Thr Thr Arg His Ser Gly Val Pro Ser Arg 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 His Tyr Ser Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 57 <211> 6 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" peptide <400> 57 Gln Asp Val Thr Thr Ala 1 5 <210> 58 <211> 3 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" peptide <400> 58 Trp Ala Thr 1 <210> 59 <211> 9 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" peptide <400> 59 Gln Gln His Tyr Ser Thr Pro Tyr Thr 1 5 <210> 60 <211> 321 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" "Polynucleotides" <400> 60 gacatccaga tgacccagtc tccaaagtct ctgtccgcct ccgtgggcga cagagtgacc 60 atcacctgta gagcctctca ggacgtgacc accgctctgg cttggtatca gcagaagcct 120 ggccagtctc ctaagctgct gatctactgg gccaccacca gacactctgg cgtgccctct 180 agattctccg gctctggctc tggcaccgac tttaccctga caatctccag cctgcagcct 240 gaggacttcg ccacctacta ctgccagcag cactacagca ccccttacac ctttggccag 300 ggcaccaagc tggaaatcaa g 321 <210> 61 <211> 447 <212> PRT <213> Mus musculus <400> 61 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Ala Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Ile Thr Trp Val Lys Gln Arg Pro Gly Gln Asp Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Gly Ser Gly Ser Thr Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Arg Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Ala Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Pro Tyr Asp Ser Asp Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro 115 120 125 Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly 130 135 140 Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn 145 150 155 160 Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr 180 185 190 Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser 195 200 205 Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro 210 215 220 Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu 245 250 255 Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro 260 265 270 Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala 275 280 285 Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val 290 295 300 Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe 305 310 315 320 Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr 325 330 335 Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu 340 345 350 Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys 355 360 365 Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn 370 375 380 Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys 405 410 415 Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly 420 425 430 Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly 435 440 445 <210> 62 <211> 1347 <212> DNA <213> Mus musculus <400> 62 caggtccaac tgcagcagcc tggggctgag cttgcgaagc ctggggcttc agtgaagatg 60 tcctgcaagg cttctggcta caccttcacc agctactgga taacctgggt gaagcagagg 120 cctggacaag accttgagtg gattggaaat atttatcctg gtagtggtag tacttactac 180 aatgagaagt tcaagagcaa ggccacactg actgtagaca catcctccag aacagcctac 240 atgcagctca gcagtctgac atctgcggac tctgcggtct attactgtgc cccctatgat 300 tccgactatg ctatggacta ctggggtcaa ggaacctcag tcaccgtctc ctcagctaaa 360 acaacagccc catcggtcta tccactggcc cctgtgtgtg gagatacaac tggctcctcg 420 gtgactctag gatgcctggt caagggttat ttccctgagc cagtgacctt gacctggaac 480 tctggttccc tgtccagtgg tgtgcacacc ttcccagctg tcctgcagtc tgacctctac 540 accctcagct caagcgtgac tgtaaccagc tcgacctggc ccagccagtc catcacctgc 600 aatgtggccc acccggcaag cagcaccaag gtggacaaga aaattgagcc cagagggccc 660 aaatcaagc cctgtcctcc atgcaaatgc ccagcaccta acctcttggg tggaccatcc 720 gtcttcatct tccctccaaa gatcaaggat gtactcatga tctccctgag ccccatagtc 780 acatgtgtag tcgttgatgt gagcgaggat gacccagatg tccagatcag ctggtttgtg 840 aacaacgtgg aagtgcacac tgctcagaca cagacgcata gagagatta caacagtact 900 ctccgggttg tcagtgccct cccacatccag caccaggact ggatgagtgg caaggagttc 960 aaatgcaagg tcaacaacaa agacctccca gcgcccatcg agagaaccat ctcaaaaccc 1020 aaagggtcag taagagctcc acaggtatat gtcttgcctc caccaga ggagatgact 1080 aagaaacagg tcactctgac ctgcatggtc acagacttca tgcctgaaga catttacgtg 1140 gagtggacca acaacgggaa aacagagcta aactacaaga acactgaacc agtcctggac 1200 tctgatggtt cttattcat gtacagcaag ctgagagtgg aagaagaa ctgggtggag 1260 agaaatagct actcctgttc agtggtccac gagggtctgc acaatcacca cacgactaag 1320 agcttctccc ggactccggg ttagtaa 1347 <210> 63 <211> 214 <212> PRT <213> Mus musculus <400> 63 Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Asn Tyr 20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile 35 40 45 Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Val Ser Gly 50 55 60 Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Tyr 65 70 75 80 Glu Asp Val Gly Ile Tyr Phe Cys Leu Gln Tyr Asp Glu Phe Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205 Phe Asn Arg Asn Glu Cys 210 <210> 64 <211> 642 <212> DNA <213> Mus musculus <400> 64 gacatcaaga tgacccagtc tccatcttcc atgtatgcat ctctaggaga gagagtcact 60 atcacttgca aggcgagtca ggacattaat aactatttaa gctggttcca gcagaaacca 120 gggaaatctc ctaagaccct gatctatcgt gcaaacagat tggtagatgg ggtcccatca 180 agggtcagtg gcagtggatc tgggcaagat tattctctca ccatcagcag cctggagtat 240 gaagatgtgg gaatttattt ttgtctacag tatgatgagt ttcctctcac gttcggtgct 300 gggaccaagc tggagctgaa aagagctgac gccgctccta ccgtgtccat ctttccacct 360 agcagcgagc agctgacaag cggcggagcc agcgtcgtgt gcttcctgaa caacttctac 420 cccaaggaca tcaacgtgaa gtggaagatc gacggcagcg agagacagaa cggcgtgctg 480 aatagctgga ccgaccagga cagcaaggac tccacctaca gcatgtccag cacactgacc 540 ctgaccaagg acgagtacga gcggcacaac agctacacat gcgaggccac acacaagacc 600 agcacaagcc ccatcgtgaa gtccttcaac cggaacgagt gc 642 <210> 65 <211> 455 <212> PRT <213> Mus musculus <400> 65 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Ile His Trp Val Lys Glu Arg Thr Glu Gln Gly Leu Glu Trp Phe 35 40 45 Gly Arg Ile Asp Pro Glu Asp Gly Glu Ser Glu Tyr Ala Pro Lys Phe 50 55 60 Gln Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Asp Ser Met Met Val Thr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Leu Thr Val Ser Ser Lys Thr Thr Pro Pro Ser Val Tyr 115 120 125 Pro Leu Ala Pro Gly Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu 130 135 140 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Ser Val Thr Val Thr Trp 145 150 155 160 Asn Ser Gly Ser Leu Ser Ser Ser Val His Thr Phe Pro Ala Leu Leu 165 170 175 Gln Ser Gly Leu Tyr Thr Met Ser Ser Ser Val Thr Val Pro Ser Ser 180 185 190 Thr Trp Pro Ser Gln Thr Val Thr Cys Ser Val Ala His Pro Ala Ser 195 200 205 Ser Thr Thr Val Asp Lys Lys Leu Glu Pro Ser Gly Pro Ile Ser Thr 210 215 220 Ile Asn Pro Cys Pro Pro Cys Lys Glu Cys His Lys Cys Pro Ala Pro 225 230 235 240 Asn Leu Glu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Asn Ile Lys 245 250 255 Asp Val Leu Met Ile Ser Leu Thr Pro Lys Val Thr Cys Val Val Val 260 265 270 Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn 275 280 285 Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr 290 295 300 Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro Ile Gln His Gln Asp 305 310 315 320 Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu 325 330 335 Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys Ile Lys Gly Leu Val Arg 340 345 350 Ala Pro Gln Val Tyr Ile Leu Pro Pro Pro Ala Glu Gln Leu Ser Arg 355 360 365 Lys Asp Val Ser Leu Thr Cys Leu Val Val Gly Phe Asn Pro Gly Asp 370 375 380 Ile Ser Val Glu Trp Thr Ser Asn Gly His Thr Glu Glu Asn Tyr Lys 385 390 395 400 Asp Thr Ala Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Ile Tyr Ser 405 410 415 Lys Leu Asn Met Lys Thr Ser Lys Trp Glu Lys Thr Asp Ser Phe Ser 420 425 430 Cys Asn Val Arg His Glu Gly Leu Lys Asn Tyr Tyr Leu Lys Lys Thr 435 440 445 Ile Ser Arg Ser Pro Gly Lys 450 455 <210> 66 <211> 1365 <212> DNA <213> Mus musculus <400> 66 gaggttcagc tgcagcagtc tggggcagaa cttgtgaagc caggggcctc agtcaagttg 60 tcctgcacag cctctggctt caacattaaa gactactata tacactgggt gaaagagagg 120 actgaacagg gcctggagtg gtttggaagg attgatcctg aggatggtga aagtgaatat 180 gccccgaaat tccagggcaa ggccacttta acagcagaca catcctccaa tacagcctac 240 ctgcagctca gcagcctgac atctgaggac actgccgtct attactgtac tagaggagac 300 tctatgatgg ttacctactt tgactactgg ggccaaggca ccactctcac ggtctcctca 360 aagaccacac ctcctagcgt gtaccctctg gctcctggct gtggcgatac aacaggcagc 420 tctgtgacac tgggctgcct ggtcaagggc tactttcctg agagcgtgac agtgacctgg 480 aacagcggca gcctgtctag cagcgtgcac acctttccag ctctgctcca gagcggcctg 540 tacaccatgt cctctagtgt gaccgtgcct agcagcacct ggcctagcca gacagtgaca 600 tgtagcgtgg cccatcctgc cagcagcaca accgtggaca agaagctgga acctagcggc 660 cccatcagca ccatcaatcc ctgtcctcca tgcaaagaat gccacaagtg ccccgctcct 720 aacctggaag gtggcccaag cgtgttcatc ttcccaccta acatcaagga cgtgctgatg 780 atcagcctga cacctaaagt gacctgcgtg gtggtggacg tgtccgagga tgatcccgat 840 gtgcagatca gttggttcgt gaacaacgtg gaagtgcaca cagcccagac acagacccac 900 agagaggact acaatagcac cattcgcgtg gtgtccacac tgcctatcca gcaccaggat 960 tggatgagcg gcaaagagtt caagtgcaaa gtgaacaaca aggacctgcc ttctccaatc 1020 gagcggacca tcagcaagat caagggactc gtcagagccc ctcaggtgta catcttgcct 1080 ccaccagccg agcagctgag cagaaaggat gtgtccctga cctgtctggt cgtgggcttc 1140 aaccctggcg acatcagcgt ggaatggacc agcaatggcc acaccgagga aaactacaag 1200 gacacagccc ctgtgctgga cagcgacggc agctacttca tctacagcaa gctgaacatg 1260 aagaccagca agtgggagaa aaccgacagc ttctcctgca acgtgcggca cgagggcctg 1320 aagaactact acctgaagaa aaccatctct cggagccccg gcaag 1365 <210> 67 <211> 214 <212> PRT <213> Mus musculus <400> 67 Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Phe Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Ser 50 55 60 Thr Gly Ser Gly Thr Asp Tyr Ala Leu Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Arg Ser Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205 Phe Asn Arg Asn Glu Cys 210 <210> 68 <211> 642 <212> DNA <213> Mus musculus <400> 68 gacattgtga tgacccagtc tcacaaattc atgtccacat cagtaggaga cagggtcagc 60 atcacctgca aggccagtca ggatgtgagt actgctgtag cctggtatca acaaaaacca 120 gggcaatctc ctaaactact gatttactgg gctttcaccc gtcacactgg agtccctgat 180 cgcttcacaa gcactggatc tgggacagat tatgctctca ccatcagcag tgtgcaggct 240 gaagacctgg cactttatta ctgtcagcaa cattatcgca gtccgtggac gttcggtgga 300 ggcaccaaac tggaaatcaa aagagctgac gccgctccta ccgtgtccat ctttccacct 360 agcagcgagc agctgacaag cggcggagcc agcgtcgtgt gcttcctgaa caacttctac 420 cccaaggaca tcaacgtgaa gtggaagatc gacggcagcg agagacagaa cggcgtgctg 480 aatagctgga ccgaccagga cagcaaggac tccacctaca gcatgtccag cacactgacc 540 ctgaccaagg acgagtacga gcggcacaac agctacacat gcgaggccac acacaagacc 600 agcacaagcc ccatcgtgaa gtccttcaac cggaacgagt gc 642 <210> 69 <211> 453 <212> PRT <213> Mus musculus <400> 69 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Glu Asp Tyr 20 25 30 Tyr Ile His Trp Val Lys Glu Arg Thr Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Glu Asp Gly Glu Thr Thr Tyr Ala Pro Gln Phe 50 55 60 Gln Gly Lys Ala Thr Ile Ile Pro Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ala Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ile Tyr Leu Asp Pro Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu 115 120 125 Ala Pro Gly Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys 130 135 140 Leu Val Lys Gly Tyr Phe Pro Glu Ser Val Thr Val Thr Trp Asn Ser 145 150 155 160 Gly Ser Leu Ser Ser Ser Val His Thr Phe Pro Ala Leu Leu Gln Ser 165 170 175 Gly Leu Tyr Thr Met Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp 180 185 190 Pro Ser Gln Thr Val Thr Cys Ser Val Ala His Pro Ala Ser Ser Thr 195 200 205 Thr Val Asp Lys Lys Leu Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn 210 215 220 Pro Cys Pro Pro Cys Lys Glu Cys His Lys Cys Pro Ala Pro Asn Leu 225 230 235 240 Glu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Asn Ile Lys Asp Val 245 250 255 Leu Met Ile Ser Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Val 260 265 270 Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val 275 280 285 Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser 290 295 300 Thr Ile Arg Val Val Ser Thr Leu Pro Ile Gln His Gln Asp Trp Met 305 310 315 320 Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ser 325 330 335 Pro Ile Glu Arg Thr Ile Ser Lys Ile Lys Gly Leu Val Arg Ala Pro 340 345 350 Gln Val Tyr Ile Leu Pro Pro Pro Ala Glu Gln Leu Ser Arg Lys Asp 355 360 365 Val Ser Leu Thr Cys Leu Val Val Gly Phe Asn Pro Gly Asp Ile Ser 370 375 380 Val Glu Trp Thr Ser Asn Gly His Thr Glu Glu Asn Tyr Lys Asp Thr 385 390 395 400 Ala Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Ile Tyr Ser Lys Leu 405 410 415 Asn Met Lys Thr Ser Lys Trp Glu Lys Thr Asp Ser Phe Ser Cys Asn 420 425 430 Val Arg His Glu Gly Leu Lys Asn Tyr Tyr Leu Lys Lys Thr Ile Ser 435 440 445 Arg Ser Pro Gly Lys 450 <210> 70 <211> 1359 <212> DNA <213> Mus musculus <400> 70 gaggttcagc tgcagcagtc tggcgccgag cttgtgaaac ctggcgcctc tgtgaagctg 60 agctgtaccg ccagcggctt caacatcgag gactactaca tccactgggt caaagagcgg 120 accgagcagg gactcgagtg gatcggaaga atcgaccccg aggacggcga gacaacatac 180 gcccctcagt ttcagggcaa agccacaatc atccccgaca ccagcagcaa caccgcctac 240 atgcaactga gcagcctgac ctctgaagat gccgccgtgt actactgcgc ccggtccatc 300 tatctggacc ccatggatta ttggggccag ggcacaagcg tgaccgtgtc ctctaagacc 360 acacctccta gcgtgtaccc tctggctcct ggctgtggcg atacaacagg cagctctgtg 420 acactgggct gcctggtcaa gggctacttt cctgagagcg tgacagtgac ctggaacagc 480 ggcagcctgt ctagcagcgt gcacacctttt ccagctctgc tccagagcgg cctgtacacc 540 atgtcctcta gtgtgaccgt gcctagcagc acctggccta gccagacagt gacatgtagc 600 gtggcccatc ctgccagcag cacaaccgtg cacaagaagc tggaacctag cggccccatc agcaccatca atccctgtcc tccatgcaaa gatgccaca agtgccccgc tcctaacctg gaaggtggcc caagcgtgtt catcttccca cctaacatca aggacgtgct gatgatcagc ctgacaccta aagtgacctg cgtggtggtg gacgtgtccg aggatgatcc cgatgtgcag 840 atcagttggt tcgtgaacaa cgtggaagtg cacacagccc agacacagac ccacagagag gactacaata gcaccattcg cgtggtgtcc acactgccta tccagcacca ggattggatg agcggcaaag agttcaagtg caaagtgaac aacaaggacc tgccttctcc aatcgagcgg accatcagca agatcaaggg actcgtcaga gccctcagg tgtacatctt gcctccacca gccgagcagc tgagcagaaa ggatgtgtcc ctgacctgtc tggtcgtggg cttcaaccct 1140 ggcgacatca gcgtggaatg gaccagcaat ggccacaccg aggaaaacta caaggacaca 1200 gcccctgtgc tggacagcga cggcagctac ttcatctaca gcaagctgaa catgaagacc 1260 agcaagtggg agaaaaccga cagcttctcc tgcaacgtgc ggcacgaggg cctgaagaac 1320 tactacctga agaaaaccat ctctcggagc cccggcaag 1359 <210> 71 <211> 214 <212> PRT <213> Mus musculus <400> 71 Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Thr Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ile Leu Ile 35 40 45 Tyr Trp Ala Thr Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Ile Ser Gly Thr Thr Tyr Ile Leu Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205 Phe Asn Arg Asn Glu Cys 210 <210> 72 <211> 642 <212> DNA <213> Mus musculus <400> 72 gacatcgtga tgacccagag ccacaagttc atgagcacca gcgtgggcga cagagtgtcc 60 atcacctgta aagccagcca ggacgtgaca acagccgtgg cctggtatca gcagaagcct 120 ggccagtctc ctaagatcct gatctactgg gccaccacca gacacaccgg cgtgccagat 180 agattcaccg gcagcatcag cggcaccacc tacatcctga caatcagctc tgtgcaggcc 240 gaggatctgg ccctgtacta ctgtcagcag cactacagca ccccttacac ctttggcgga 300 ggcaccaagc tggaaatcaa gagagctgac gccgctccta ccgtgtccat ctttccacct 360 agcagcgagc agctgacaag cggcggagcc agcgtcgtgt gcttcctgaa caacttctac 420 cccaaggaca tcaacgtgaa gtggaagatc gacggcagcg agagacagaa cggcgtgctg 480 aatagctgga ccgaccagga cagcaaggac tccacctaca gcatgtccag cacactgacc 540 ctgaccaagg acgagtacga gcggcacaac agctacacat gcgaggccac acacaagacc 600 agcacaagcc ccatcgtgaa gtccttcaac cggaacgagt gc 642 <210> 73 <211> 447 <212> PRT <213> Artificial Sequence <220> <221> source <223> / Comment=“Description of artificial sequence: synthetic polypeptide” <400> 73 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Gly Ser Gly Ser Thr Tyr Tyr Asn Glu Lys Phe 50 55 60 Lys Ser Lys Ala Thr Ile Thr Arg Asp Thr Ser Ser Arg Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Pro Tyr Asp Ala Asp Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 10,5 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu [[ID=ll]]340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp A385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 <210> 74 [[ID=:39]]<211> 1341 <212> DNA <213> Artificial sequence <220> <221> source <223> / Comment=“Description of artificial sequence: synthetic polynucleotide” <400> 74 gaagtgcagc tggtgcaatc tggcgccgaa gtgaagaaac ctggcgcctc tgtgaaggtg 60 tcctgcaagg cttccggcta cacctttacc agctactgga tcacctgggt ccgacaggct 120 cctggccaga gactggaatg gatcggcaac atctaccctg gctccggctc cacctactac 180 aacgagaagt tcaagtccaa ggccacaatc acccgggaca cctcttccag aaccgcctac 240 atggaactgt ccagcctgag atctgaggac accgccgtgt actactgcgc cccttacgac 300 gccgactacg ccatggatta ttggggccag ggcaccctgg tcaccgtgtc ctctgcttct 360 accaagggac ccagcgtgtt ccctctggct ccttccagca agtctacctc tggcggaaca 420 gctgctctgg gctgcctggt caaggactac tttcctgagc ctgtgaccgt gtcttggaac 480 tctggcgctc tgacatccgg cgtgcacaca tttccagctg tgctgcagtc ctccggcctg 540 tactctctgt cctctgtcgt gaccgtgcct tcctctagcc tgggcaccca gacctacatc 600 tgcaatgtga accacaagcc ttccaacacc aaggtggaca agaaggtgga acccaagtcc 660 tgcgacaaga cccacacctg tcctccatgt cctgctccag aactgctcgg cggaccttcc 720 gtgttcctgt ttcctccaaa gcctaaggac accctgatga tctctcggac ccctgaagtg 780 acctgcgtgg tggtggatgt gtctcacgag gacccagaag tgaagttcaa ttggtacgtg 840 gacggcgtgg aagtgcacaa cgccaagacc aagcctagag aggaacagta cgccagcacc 900 tacagagtgg tgtccgtgct gacagtgctg caccaggatt ggctgaacgg caaagagtac 960 aagtgcaagg tgtccaacaa ggccctgcct gctcctatcg aaaagaccat cagcaaggcc 1020 aagggccagc ctagagaacc ccaggtttac accttgcctc catctcggga cgagctgacc 1080 aagaaccagg tgtccctgac ctgtctcgtg aagggcttct acccctccga catcgccgtg 1140 gaatgggagt ctaatggcca gccagagaac aactacaaga caacccctcc tgtgctggac 1200 tccgacggct cattcttcct gtactccaag ctgaccgtgg acaagtccag atggcagcag 1260 ggcaacgtgt tctcctgctc cgtgatgcac gaggccctgc acaatcacta cacacagaag 1320 tccctgtctc tgtcccctgg c 1341 <210> 75 <211> 214 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" "Polypeptide" <400> 75 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 Lys Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Arg Ala Asn Arg Leu Val Glu Gly Val Pro Ser Arg 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 Phe Cys Leu Gln Tyr Asp Glu Phe Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 76 <211> 642 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" "Polynucleotides" <400> 76 gacatccaga tgacccagtc tccatcctct ctgtccgcct ctgtgggcga cagagtgacc 60 atcacatgca aggccagcca ggacatctcc aactacctgt cctggttcca gcagaagcct 120 ggcaaggctc ccaagctgct gatctacaga gccaacagac tggtggaagg cgtgccctcc 180 ggcaaggctc ccaagctgct gatctacaga gccaacagac tggtggaagg cgtgccctcc 180 agattctccg gatctggctc tggcaccgac tttaccctga caatctccag cctgcagcct 240 agattctccg gatctggctc tggcaccgac tttaccctga caatctccag cctgcagcct 240 gaggacttcg ctacctactt ctgcctgcaa tacgacgagt tccctctgac ctttggcgga 300 gaggacttcg ctacctactt ctgcctgcaa tacgacgagt tccctctgac ctttggcgga 300 ggcaccaagg tggaaatcaa gcggacagtg gccgctcctt ccgtgttcat cttcccacct 360 ggcaccaagg tggaaatcaa gcggacagtg gccgctcctt ccgtgttcat cttcccacct 360 tccgacgagc agctgaagtc cggcacagct tctgtcgtgt gcctgctgaa caacttctac 420 tccgacgagc agctgaagtc cggcacagct tctgtcgtgt gcctgctgaa caacttctac 420 cctcgggaag ccaaggtgca gtggaaggtg gacaatgccc tgcagtccgg caactcccaa 480 cctcgggaag ccaaggtgca gtggaaggtg gacaatgccc tgcagtccgg caactcccaa 480 gagtctgtga ccgagcagga ctccaaggac agcacctaca gcctgtcctc cacactgacc 540 gagtctgtga ccgagcagga ctccaaggac agcacctaca gcctgtcctc cacactgacc 540 ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccatcagggc 600 ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccatcagggc 600 ctgtctagcc ctgtgaccaa gtctttcaac cggggcgagt gt 642 ctgtctagcc ctgtgaccaa gtctttcaac cggggcgagt gt 642 <210> 77<210> 77 <211> 449<211> 449 <212> PRT<212> PRT <213> 人工序列 <213> Artificial sequence <220> <220> 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Glu Asp Ala Glu Ser Glu Tyr Ala Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Asp Ser Met Met Val Thr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly <210> 78 <211> 1347 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" "Polynucleotides" <400> 78 gaagtgcagc tggtgcaatc tggcgccgaa gtgaagaaac ctggcgcctc tgtgaaggtg 60 tcctgcaagg cctctggctt caacatcaag gactactaca tccactgggt ccgacaggct 120 accggacagg gacttgagtg gatgggcaga atcgaccctg aggacgccga gtctgagtac 180 gcccctaagt ttcagggcag agtgaccatc accgccgaca cctctaccga caccgcctac 240 atggaactgt ccagcctgag atctgaggac accgccgtgt actactgcac cagaggcgac 300 tccatgatgg ttacctactt cgactactgg ggccagggca ccctggtcac agtttcttcc 360 gcttccacca agggacccag cgtgttccct ctggctcctt ccagcaagtc tacctctggc 420 ggaacagctg ctctgggctg cctggtcaag gattacttcc ctgagcctgt gaccgtgtcc 480 tggaactctg gcgctctgac atccggcgtg cacacctttc cagctgtgct gcaatcctcc 540 ggcctgtact ctctgtcctc cgtcgtgacc gtgccttcta gctctctggg cacccagacc 600 tacatctgca atgtgaacca caagccttcc aacaccaagg tggacaagaa ggtggaaccc 660 aagtcctgcg acaagaccca cacctgtcct ccatgtcctg ctccagaact gctcggcgga 720 ccttccgtgt tcctgtttcc tccaaagcct aaggacaccc tgatgatctc tcggacccct 780 gaagtgacct gcgtggtggt ggatgtgtct cacgaggacc cagaagtgaa gttcaattgg 840 tacgtggacg gcgtggaagt gcacaacgcc aagaccaagc ctagagagga acagtacgcc 900 tccacctaca gagtggtgtc cgtgctgaca gtgctgcacc aggattggct gaacggcaaa 960 gagtacaagt gcaaggtgtc caacaaggca ctgcccgctc ctatcgaaaa gaccatctcc 1020 aaggccaagg gccagcctag agaaccccag gtttacacct tgcctccatc tcgggacgag 1080 ctgaccaaga accaggtgtc cctgacctgt ctcgtgaagg gcttctaccc ctccgacatc 1140 gccgtggaat gggagtctaa tggccagcca gagaacaact acaagacaac ccctcctgtg 1200 ctggactccg acggctcatt cttcctgtac tccaagctga ccgtggacaa gtccagatgg 1260 cagcagggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa tcactacaca 1320 cagaagtctc tgtccctgtc tcctggc 1347 <210> 79 <211> 214 <212> PRT <213> Artificial Sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" "Polypeptide" <400> 79 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 Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Phe Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ala Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Arg Ser Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 80 <211> 642 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" "Polynucleotides" <400> 80 gacatccaga tgacccagtc tccatcctct ctgtccgcct ctgtgggcga cagagtgacc 60 atcacatgca aggcctctca ggacgtgtcc accgccgttg cttggtatca gcagaagcct 120 ggcaaggccc ctaagctgct gatctactgg gccttcacca gacacaccgg cgtgccctct 180 aggttctccg gctctggctc tggcaccgat tacgctctga caatctccag cctgcagcct 240 gaggacttcg ccacctacta ctgccagcag cactacagaa gcccctggac atttggcgga 300 ggcaccaagg tggaaatcaa gcggacagtg gccgctcctt ccgtgttcat cttcccacct 360 tccgacgagc agctgaagtc cggcacagct tctgtcgtgt gcctgctgaa caacttctac 420 cctcgggaag ccaaggtgca gtggaaggtg gacaatgccc tgcagtccgg caactcccaa 480 gagtctgtga ccgagcagga ctccaaggac agcacctaca gcctgtcctc cacactgacc 540 ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccatcagggc 600 ctgtctagcc ctgtgaccaa gtctttcaac cggggcgagt gt 642 <210> 81 <211> 447 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of artificial sequence: Synthetic polypeptide" <400> 81 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Glu Asp Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Glu Asp Ala Glu Thr Thr Tyr Ser Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Ile Pro Asp Thr Ser Ala Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ile Tyr Leu Asp Pro Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 <210> 82 <211> 1341 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" "Polynucleotides" <400> 82 caggtgcagc tggtgcagtc tggcgccgaa gtgaaaaagc ctggcgcctc tgtgaaggtg 60 tcctgcaagg cctctggctt caacatcgag gactactaca tgcactgggt ccgacaggcc 120 cctggccaga gattggaatg gatgggcaga atcgaccccg aggacgccga gacaacctac 180 tctcctaagt tccagggccg cgtgacaatc atccctgaca cctctgccaa caccgcctac 240 atggaactgt ccagcctgag atctgaggac accgccgtgt actactgcgc ccggtctatc 300 tacctggacc ctatggacta ttggggccag ggcaccctgg tcacagtgtc ctctgcttct 360 accaagggac ccagcgtgtt ccctctggct ccttccagca agtctacctc tggcggaaca 420 gctgctctgg gctgcctggt caaggactac tttccagagc ctgtgaccgt gtcctggaac 480 tctggcgctc tgacatctgg cgtgcacacc tttccagctg tgctgcagtc ctccggcctg 540 tactctctgt cctctgtcgt gaccgtgcct tccagctctc tgggaaccca gacctacatc 600 tgcaatgtga accacaagcc ttccaacacc aaggtggaca agaaggtgga acccaagtcc 660 tgcgacaaga cccacacctg tcctccatgt cctgctccag aagctgctgg cggcccttcc 720 gtgtttctgt tccctccaaa gcctaaggac accctgatga tctctcggac ccctgaagtg 780 acctgcgtgg tggtggatgt gtctcacgag gacccagaag tgaagttcaa ttggtacgtg 840 gacggcgtgg aagtgcacaa cgccaagacc aagcctagag aggaacagta caactccacc 900 tacagagtgg tgtccgtgct gaccgtgctg caccaggatt ggctgaacgg caaagagtac 960 aagtgcaagg tgtccaacaa ggcactgccc gctcctatcg aaaagaccat ctccaaggcc 1020 aagggccagc ctagggaacc ccaggtttac accctgcctc caagccggga agagatgacc 1080 aagaaccagg tgtccctgac ctgcctcgtg aagggcttct acccttccga catcgccgtg 1140 gaatgggaga gcaatggcca gccagagaac aactacaaga caacccctcc tgtgctggac 1200 tccgacggct cattcttcct gtactccaag ctgacagtgg acaagtccag atggcagcag 1260 ggcaacgtgt tctcctgctc cgtgatgcac gaggccctgc acaatcacta cacacagaag 1320 tccctgtctc tgtcccctgg c 1341 <210> 83 <211> 214 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of artificial sequence: Synthetic polypeptide" <400> 83 Asp Ile Gln Met Thr Gln Ser Pro Lys Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Thr Thr Ala 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Thr Thr Arg His Ser Gly Val Pro Ser Arg 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 His Tyr Ser Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 84 <211> 642 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of artificial sequence: synthetic polynucleotide" <400> 84 gacatccaga tgacccagtc tccaaagtct ctgtccgcct ccgtgggcga cagagtgacc 60 atcacctgta gagcctctca ggacgtgacc accgctctgg cttggtatca gcagaagcct 120 ggccagtctc ctaagctgct gatctactgg gccaccacca gacactctgg cgtgccctct 180 agattctccg gctctggctc tggcaccgac tttaccctga caatctccag cctgcagcct 240 gaggacttcg ccacctacta ctgccagcag cactacagca ccccttacac ctttggccag 300 ggcaccaagc tggaaatcaa gcggacagtg gccgctcctt ccgtgttcat cttcccacct 360 tccgacgagc agctgaagtc cggcacagct tctgtcgtgt gcctgctgaa caacttctac 420 cctcgggaag ccaaggtgca gtggaaggtg gacaatgccc tgcagtccgg caactcccaa 480 gagtctgtga ccgagcagga ctccaaggac agcacctaca gcctgtcctc cacactgacc 540 ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccatcagggc 600 ctgtctagcc ctgtgaccaa gtctttcaac cggggcgagt gt 642 <210> 85 <211> twenty one <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" Introduction <400> 85 aagcagggca gacattgcga t 21 <210> 86 <211> twenty one <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" Introduction <400> 86 caggatgtgg ctctaggcta t 21 <210> 87 <211> 20 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" Introduction <400> 87 acccacactg tgcccatcta 20 <210> 88 <211> 20 <212> DNA <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" Introduction <400> 88 cacgctcggt caggatcttc 20 <210> 89 <211> 118 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" "Polypeptide" <400> 89 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Glu Asp Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Glu Asp Gly Glu Thr Thr Tyr Ser Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Ile Pro Asp Thr Ser Ala Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ile Tyr Leu Asp Pro Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 90 <211> 118 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" "Polypeptide" <400> 90 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Thr Ala Ser Gly Phe Asn Ile Glu Asp Tyr 20 25 30 Tyr Ile Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Glu Asp Gly Glu Thr Gly Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asn Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ile Tyr Leu Asp Pro Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 91 <211> 118 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" "Polypeptide" <400> 91 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Glu Asp Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Glu Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Glu Asp Gly Glu Thr Thr Tyr Ala Pro Lys Phe 50 55 60 Gln Gly Arg Val Thr Ser Thr Arg Asp Thr Ser Ile Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Val Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ile Tyr Leu Asp Pro Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 92 <211> 118 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" "Polypeptide" <400> 92 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Glu Asp Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Glu Gln Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asp Pro Glu Asp Gly Glu Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ile Tyr Leu Asp Pro Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 93 <211> 107 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" "Polypeptide" <400> 93 Asp Ile Gln Met Thr Gln Ser Pro Lys Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Thr Thr Ala 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Thr Thr Arg His Ser Gly Val Pro Ser Arg 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 His Tyr Ser Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 94 <211> 107 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" "Polypeptide" <400> 94 Asp Ile Gln Met Thr Gln Ser Pro Lys Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Thr Thr Ala 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Thr Ser Leu Gln Ser Gly Val Pro Ser Arg 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 His Tyr Ser Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 95 <211> 107 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" "Polypeptide" <400> 95 Asp Ile Gln Met Thr Gln Ser Pro Lys Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Val Thr Thr Ala 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Thr Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 96 <211> 107 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" "Polypeptide" <400> 96 Asp Ile Val Met Thr Gln Ser Pro Lys Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Asp Val Thr Thr Ala 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Thr Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 97 <211> 808 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" "Polypeptide" <400> 97 Met Pro Val Ala Glu Ala Pro Gln Val Ala Gly Gly Gln Gly Asp Gly 1 5 10 15 Gly Asp Gly Glu Glu Ala Glu Pro Glu Gly Met Phe Lys Ala Cys Glu 20 25 30 Asp Ser Lys Arg Lys Ala Arg Gly Tyr Leu Arg Leu Val Pro Leu Phe 35 40 45 Val Leu Leu Ala Leu Leu Val Leu Ala Ser Ala Gly Val Leu Leu Trp 50 55 60 Tyr Phe Leu Gly Tyr Lys Ala Glu Val Met Val Ser Gln Val Tyr Ser 65 70 75 80 Gly Ser Leu Arg Val Leu Asn Arg His Phe Ser Gln Asp Leu Thr Arg 85 90 95 Arg Glu Ser Ser Ala Phe Arg Ser Glu Thr Ala Lys Ala Gln Lys Met 100 105 110 Leu Lys Glu Leu Ile Thr Ser Thr Arg Leu Gly Thr Tyr Tyr Asn Ser 115 120 125 Ser Ser Val Tyr Ser Phe Gly Glu Gly Pro Leu Thr Cys Phe Phe Trp 130 135 140 Phe Ile Leu Gln Ile Pro Glu His Arg Arg Leu Met Leu Ser Pro Glu 145 150 155 160 Val Val Gln Ala Leu Leu Val Glu Glu Leu Leu Ser Thr Val Asn Ser 165 170 175 Ser Ala Ala Val Pro Tyr Arg Ala Glu Tyr Glu Val Asp Pro Glu Gly 180 185 190 Leu Val Ile Leu Glu Ala Ser Val Lys Asp Ile Ala Ala Leu Asn Ser 195 200 205 Thr Leu Gly Cys Tyr Arg Tyr Ser Tyr Val Gly Gln Gly Gln Val Leu 210 215 220 Arg Leu Lys Gly Pro Asp His Leu Ala Ser Ser Cys Leu Trp His Leu 225 230 235 240 Gln Gly Pro Lys Asp Leu Met Leu Lys Leu Arg Leu Glu Trp Thr Leu 245 250 255 Ala Glu Cys Arg Asp Arg Leu Ala Met Tyr Asp Val Ala Gly Pro Leu 260 265 270 Glu Lys Arg Leu Ile Thr Ser Val Tyr Gly Cys Ser Arg Gln Glu Pro 275 280 285 Val Val Glu Val Leu Ala Ser Gly Ala Ile Met Ala Val Val Trp Lys 290 295 300 Lys Gly Leu His Ser Tyr Tyr Asp Pro Phe Val Leu Ser Val Gln Pro 305 310 315 320 Val Val Phe Gln Ala Cys Glu Val Asn Leu Thr Leu Asp Asn Arg Leu 325 330 335 Asp Ser Gln Gly Val Leu Ser Thr Pro Tyr Phe Pro Ser Tyr Tyr Ser 340 345 350 Pro Gln Thr His Cys Ser Trp His Leu Thr Val Pro Ser Leu Asp Tyr 355 360 365 Gly Leu Ala Leu Trp Phe Asp Ala Tyr Ala Leu Arg Arg Gln Lys Tyr 370 375 380 Asp Leu Pro Cys Thr Gln Gly Gln Trp Thr Ile Gln Asn Arg Arg Leu 385 390 395 400 Cys Gly Leu Arg Ile Leu Gln Pro Tyr Ala Glu Arg Ile Pro Val Val 405 410 415 Ala Thr Ala Gly Ile Thr Ile Asn Phe Thr Ser Gln Ile Ser Leu Thr 420 425 430 Gly Pro Gly Val Arg Val His Tyr Gly Leu Tyr Asn Gln Ser Asp Pro 435 440 445 Cys Pro Gly Glu Phe Leu Cys Ser Val Asn Gly Leu Cys Val Pro Ala 450 455 460 Cys Asp Gly Val Lys Asp Cys Pro Asn Gly Leu Asp Glu Arg Asn Cys 465 470 475 480 Val Cys Arg Ala Thr Phe Gln Cys Lys Glu Asp Ser Thr Cys Ile Ser 485 490 495 Leu Pro Lys Val Cys Asp Gly Gln Pro Asp Cys Leu Asn Gly Ser Asp 500 505 510 Glu Glu Gln Cys Gln Glu Gly Val Pro Cys Gly Thr Phe Thr Phe Gln 515 520 525 Cys Glu Asp Arg Ser Cys Val Lys Lys Pro Asn Pro Gln Cys Asp Gly 530 535 540 Arg Pro Asp Cys Arg Asp Gly Ser Asp Glu Glu His Cys Asp Cys Gly 545 550 555 560 Leu Gln Gly Pro Ser Ser Arg Ile Val Gly Gly Ala Val Ser Ser Glu 565 570 575 Gly Glu Trp Pro Trp Gln Ala Ser Leu Gln Val Arg Gly Arg His Ile 580 585 590 Cys Gly Gly Ala Leu Ile Ala Asp Arg Trp Val Ile Thr Ala Ala His 595 600 605 Cys Phe Gln Glu Asp Ser Met Ala Ser Thr Val Leu Trp Thr Val Phe 610 615 620 Leu Gly Lys Val Trp Gln Asn Ser Arg Trp Pro Gly Glu Val Ser Phe 625 630 635 640 Lys Val Ser Arg Leu Leu Leu His Pro Tyr His Glu Glu Asp Ser His 645 650 655 Asp Tyr Asp Val Ala Leu Leu Gln Leu Asp His Pro Val Val Arg Ser 660 665 670 Ala Ala Val Arg Pro Val Cys Leu Pro Ala Arg Ser His Phe Phe Glu 675 680 685 Pro Gly Leu His Cys Trp Ile Thr Gly Trp Gly Ala Leu Arg Glu Gly 690 695 700 Gly Pro Ile Ser Asn Ala Leu Gln Lys Val Asp Val Gln Leu Ile Pro 705 710 715 720 Gln Asp Leu Cys Ser Glu Val Tyr Arg Tyr Gln Val Thr Pro Arg Met 725 730 735 Leu Cys Ala Gly Tyr Arg Lys Gly Lys Lys Asp Ala Cys Gln Gly Asp 740 745 750 Ser Gly Gly Pro Leu Val Cys Lys Ala Leu Ser Gly Arg Trp Phe Leu 755 760 765 Ala Gly Leu Val Ser Trp Gly Leu Gly Cys Gly Arg Pro Asn Tyr Phe 770 775 780 Gly Val Tyr Thr Arg Ile Thr Gly Val Ile Ser Trp Ile Gln Gln Val 785 790 795 800 Val Thr His His His His His 805 <210> 98 <211> 817 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" "Polypeptide" <400> 98 Met Pro Arg Cys Phe Gln Leu Pro Cys Ser Thr Arg Met Pro Thr Thr 1 5 10 15 Glu Val Pro Gln Ala Ala Asp Gly Gln Gly Asp Ala Gly Asp Gly Glu 20 25 30 Glu Ala Ala Glu Pro Glu Gly Lys Phe Lys Pro Pro Lys Asn Thr Lys 35 40 45 Arg Lys Asn Arg Asp Tyr Val Arg Phe Thr Pro Leu Leu Leu Val Leu 50 55 60 Ala Ala Leu Val Ser Ala Gly Val Met Leu Trp Tyr Phe Leu Gly Tyr 65 70 75 80 Lys Ala Glu Val Thr Val Ser Gln Val Tyr Ser Gly Ser Leu Arg Val 85 90 95 Leu Asn Arg His Phe Ser Gln Asp Leu Gly Arg Arg Glu Ser Ile Ala 100 105 110 Phe Arg Ser Glu Ser Ala Lys Ala Gln Lys Met Leu Gln Glu Leu Val 115 120 125 Ala Ser Thr Arg Leu Gly Thr Tyr Tyr Asn Ser Ser Ser Val Tyr Ser 130 135 140 Phe Gly Glu Gly Pro Leu Thr Cys Phe Phe Trp Phe Ile Leu Asp Ile 145 150 155 160 Pro Glu Tyr Gln Arg Leu Thr Leu Ser Pro Glu Val Val Arg Glu Leu 165 170 175 Leu Val Asp Glu Leu Leu Ser Asn Ser Ser Thr Leu Ala Ser Tyr Lys 180 185 190 Thr Glu Tyr Glu Val Asp Pro Glu Gly Leu Val Ile Leu Glu Ala Ser 195 200 205 Val Asn Asp Ile Val Val Leu Asn Ser Thr Leu Gly Cys Tyr Arg Tyr 210 215 220 Ser Tyr Val Asn Pro Gly Gln Val Leu Pro Leu Lys Gly Pro Asp Gln 225 230 235 240 Gln Thr Thr Ser Cys Leu Trp His Leu Gln Gly Pro Glu Asp Leu Met 245 250 255 Ile Lys Val Arg Leu Glu Trp Thr Arg Val Asp Cys Arg Asp Arg Val 260 265 270 Ala Met Tyr Asp Ala Ala Gly Pro Leu Glu Lys Arg Leu Ile Thr Ser 275 280 285 Val Tyr Gly Cys Ser Arg Gln Glu Pro Val Met Glu Val Leu Ala Ser 290 295 300 Gly Ser Val Met Ala Val Val Trp Lys Lys Gly Met His Ser Tyr Tyr 305 310 315 320 Asp Pro Phe Leu Leu Ser Val Lys Ser Val Ala Phe Gln Asp Cys Gln 325 330 335 Val Asn Leu Thr Leu Glu Gly Arg Leu Asp Thr Gln Gly Phe Leu Arg 340 345 350 Thr Pro Tyr Tyr Pro Ser Tyr Tyr Ser Pro Ser Thr His Cys Ser Trp 355 360 365 His Leu Thr Val Pro Ser Leu Asp Tyr Gly Leu Ala Leu Trp Phe Asp 370 375 380 Ala Tyr Ala Leu Arg Arg Gln Lys Tyr Asn Arg Leu Cys Thr Gln Gly 385 390 395 400 Gln Trp Met Ile Gln Asn Arg Arg Leu Cys Gly Phe Arg Thr Leu Gln 405 410 415 Pro Tyr Ala Glu Arg Ile Pro Met Val Ala Ser Asp Gly Val Thr Ile 420 425 430 Asn Phe Thr Ser Gln Ile Ser Leu Thr Gly Pro Gly Val Gln Val Tyr 435 440 445 Tyr Ser Leu Tyr Asn Gln Ser Asp Pro Cys Pro Gly Glu Phe Leu Cys 450 455 460 Ser Val Asn Gly Leu Cys Val Pro Ala Cys Asp Gly Ile Lys Asp Cys 465 470 475 480 Pro Asn Gly Leu Asp Glu Arg Asn Cys Val Cys Arg Ala Met Phe Gln 485 490 495 Cys Gln Glu Asp Ser Thr Cys Ile Ser Leu Pro Arg Val Cys Asp Arg 500 505 510 Gln Pro Asp Cys Leu Asn Gly Ser Asp Glu Glu Gln Cys Gln Glu Gly 515 520 525 Val Pro Cys Gly Thr Phe Thr Phe Gln Cys Glu Asp Arg Ser Cys Val 530 535 540 Lys Lys Pro Asn Pro Glu Cys Asp Gly Gln Ser Asp Cys Arg Asp Gly 545 550 555 560 Ser Asp Glu Gln His Cys Asp Cys Gly Leu Gln Gly Leu Ser Ser Arg 565 570 575 Ile Val Gly Gly Thr Val Ser Ser Glu Gly Glu Trp Pro Trp Gln Ala 580 585 590 Ser Leu Gln Ile Arg Gly Arg His Ile Cys Gly Gly Ala Leu Ile Ala 595 600 605 Asp Arg Trp Val Ile Thr Ala Ala His Cys Phe Gln Glu Asp Ser Met 610 615 620 Ala Ser Pro Lys Leu Trp Thr Val Phe Leu Gly Lys Met Arg Gln Asn 625 630 635 640 Ser Arg Trp Pro Gly Glu Val Ser Phe Lys Val Ser Arg Leu Phe Leu 645 650 655 His Pro Tyr His Glu Glu Asp Ser His Asp Tyr Asp Val Ala Leu Leu 660 665 670 Gln Leu Asp His Pro Val Val Tyr Ser Ala Thr Val Arg Pro Val Cys 675 680 685 Leu Pro Ala Arg Ser His Phe Phe Glu Pro Gly Gln His Cys Trp Ile 690 695 700 Thr Gly Trp Gly Ala Gln Arg Glu Gly Gly Pro Val Ser Asn Thr Leu 705 710 715 720 Gln Lys Val Asp Val Gln Leu Val Pro Gln Asp Leu Cys Ser Glu Ala 725 730 735 Tyr Arg Tyr Gln Val Ser Pro Arg Met Leu Cys Ala Gly Tyr Arg Lys 740 745 750 Gly Lys Lys Asp Ala Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys 755 760 765 Arg Glu Pro Ser Gly Arg Trp Phe Leu Ala Gly Leu Val Ser Trp Gly 770 775 780 Leu Gly Cys Gly Arg Pro Asn Phe Phe Gly Val Tyr Thr Arg Val Thr 785 790 795 800 Arg Val Ile Asn Trp Ile Gln Gln Val Leu Thr His His His His 805 810 815 His <210> 99 <211> 806 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" "Polypeptide" <400> 99 Met Pro Val Ala Lys Ala Pro Gln Val Ala Gly Gly Gln Gly Asp Gly 1 5 10 15 Gly Asp Gly Glu Glu Ala Glu Pro Glu Gly Met Phe Glu Ala Cys Glu 20 25 30 Asp Ser Lys Arg Lys Ala Arg Gly Tyr Leu Arg Leu Ala Pro Leu Trp 35 40 45 Leu Thr Leu Val Val Leu Thr Ser Val Gly Val Leu Leu Trp Tyr Phe 50 55 60 Leu Gly Tyr Lys Ala Glu Val Thr Val Ser Gln Val Tyr Ser Gly Ser 65 70 75 80 Leu Arg Val Leu Asn Arg His Phe Ser Gln Asp Leu Thr Arg Arg Glu 85 90 95 Ser Ser Ala Phe Arg Ser Glu Thr Ala Lys Ala Gln Lys Met Leu Lys 100 105 110 Glu Leu Ile Ala Ser Thr Arg Leu Gly Thr Tyr Tyr Asn Ser Ser Ser 115 120 125 Val Tyr Ser Phe Gly Glu Gly Pro Leu Thr Cys Phe Phe Trp Phe Ile 130 135 140 Leu Gln Ile Pro Glu His Arg Arg Leu Met Leu Ser Pro Glu Val Val 145 150 155 160 Gln Ala Leu Leu Val Glu Glu Leu Leu Ser Thr Val Asn Ser Ser Ala 165 170 175 Ala Val Pro Tyr Arg Ala Glu Tyr Glu Val Asp Pro Glu Gly Leu Val 180 185 190 Ile Leu Glu Ala Ser Val Lys Asp Ile Ala Ala Leu Asn Ser Thr Leu 195 200 205 Gly Cys Tyr Arg Tyr Ser Tyr Val Gly Gln Gly Gln Val Leu Arg Leu 210 215 220 Lys Gly Pro Asp His Leu Ala Ser Ser Cys Leu Trp His Leu Gln Gly 225 230 235 240 Pro Glu Asp Leu Met Leu Lys Leu Arg Leu Glu Trp Thr Leu Ala Glu 245 250 255 Cys Arg Asp Arg Leu Ala Met Tyr Asp Val Ala Gly Pro Leu Glu Lys 260 265 270 Arg Leu Ile Thr Ser Val Tyr Gly Cys Ser Arg Gln Glu Pro Val Val 275 280 285 Glu Val Leu Ala Ser Gly Ala Ile Met Ala Val Val Trp Lys Lys Gly 290 295 300 Leu His Ser Tyr Tyr Asp Pro Phe Met Leu Ser Val Gln Ser Val Val 305 310 315 320 Phe Gln Ala Cys Glu Val Asn Leu Thr Leu Asp Asp Arg Leu Asp Ser 325 330 335 Gln Gly Val Leu Ser Thr Pro Tyr Phe Pro Ser Tyr Tyr Ser Pro Arg 340 345 350 Thr His Cys Ser Trp His Leu Thr Val Pro Ser Leu Asp Tyr Gly Leu 355 360 365 Ala Leu Trp Phe Asp Ala Tyr Ala Leu Arg Arg Gln Lys Tyr Asp Leu 370 375 380 Pro Cys Thr Gln Gly Gln Trp Thr Ile Gln Asn Arg Arg Leu Cys Gly 385 390 395 400 Leu Arg Ile Leu Gln Pro Tyr Ala Glu Arg Ile Pro Val Val Ala Thr 405 410 415 Ala Gly Ile Thr Ile Asn Phe Thr Ser Gln Ile Ser Leu Thr Gly Pro 420 425 430 Gly Val Arg Val His Tyr Gly Leu Tyr Asn Gln Ser Asp Pro Cys Pro 435 440 445 Gly Glu Phe Leu Cys Ser Val Asn Gly Leu Cys Val Pro Ala Cys Asp 450 455 460 Gly Val Lys Asp Cys Pro Asn Gly Leu Asp Glu Arg Asn Cys Val Cys 465 470 475 480 Arg Ala Thr Phe Gln Cys Gln Glu Asp Ser Thr Cys Ile Ser Leu Leu 485 490 495 Lys Val Cys Asp Gly Gln Pro Asp Cys Leu Asn Gly Ser Asp Glu Glu 500 505 510 Arg Cys Gln Glu Gly Val Pro Cys Gly Thr Phe Thr Phe Gln Cys Glu 515 520 525 Asp Gln Ser Cys Val Lys Lys Pro Asn Pro Gln Cys Asp Gly Arg Pro 530 535 540 Asp Cys Arg Asp Gly Ser Asp Glu Gln His Cys Asp Cys Gly Leu Gln 545 550 555 560 Gly Pro Ser Ser Arg Ile Val Gly Gly Ala Val Ser Ser Glu Gly Glu 565 570 575 Trp Pro Trp Gln Ala Ser Leu Gln Val Arg Gly Arg His Ile Cys Gly 580 585 590 Gly Ala Leu Ile Ala Asp Arg Trp Val Ile Thr Ala Ala His Cys Phe 595 600 605 Gln Glu Asp Ser Met Ala Ser Pro Ala Leu Trp Thr Val Phe Leu Gly 610 615 620 Lys Val Trp Gln Asn Ser Arg Trp Pro Gly Glu Val Ser Phe Lys Val 625 630 635 640 Ser Arg Leu Leu Leu His Pro Tyr His Glu Glu Asp Ser His Asp Tyr 645 650 655 Asp Val Ala Leu Leu Gln Leu Asp His Pro Val Val Arg Ser Ala Ala 660 665 670 Val Arg Pro Val Cys Leu Pro Ala Arg Ser His Phe Phe Glu Pro Gly 675 680 685 Leu His Cys Trp Ile Thr Gly Trp Gly Ala Leu Arg Glu Gly Gly Pro 690 695 700 Thr Ser Asn Ala Leu Gln Lys Val Asp Val Gln Leu Ile Pro Gln Asp 705 710 715 720 Leu Cys Ser Glu Ala Tyr Arg Tyr Gln Val Thr Pro Arg Met Leu Cys 725 730 735 Ala Gly Tyr Arg Lys Gly Lys Lys Asp Ala Cys Gln Gly Asp Ser Gly 740 745 750 Gly Pro Leu Val Cys Lys Ala Leu Ser Gly Arg Trp Phe Leu Ala Gly 755 760 765 Leu Val Ser Trp Gly Leu Gly Cys Gly Arg Pro Asn Tyr Phe Gly Val 770 775 780 Tyr Thr Arg Ile Thr Gly Val Ile Gly Trp Ile Gln Gln Val Val Thr 785 790 795 800 His His His His His His 805 <210> 100 <211> 860 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" "Polypeptide" <400> 100 Met Gly Ser Asp Arg Ala Arg Lys Gly Gly Gly Gly Pro Lys Asp Phe 1 5 10 15 Gly Ala Gly Leu Lys Tyr Asn Ser Arg His Glu Lys Val Asn Gly Leu 20 25 30 Glu Glu Gly Val Glu Phe Leu Pro Val Asn Asn Val Lys Lys Val Glu 35 40 45 Lys His Gly Pro Gly Arg Trp Val Val Leu Ala Ala Val Leu Ile Gly 50 55 60 Leu Leu Leu Val Leu Leu Gly Ile Gly Phe Leu Val Trp His Leu Gln 65 70 75 80 Tyr Arg Asp Val Arg Val Gln Lys Val Phe Asn Gly Tyr Met Arg Ile 85 90 95 Thr Asn Glu Asn Phe Val Asp Ala Tyr Glu Asn Ser Asn Ser Thr Glu 100 105 110 Phe Val Ser Leu Ala Ser Lys Val Lys Asp Ala Leu Lys Leu Leu Tyr 115 120 125 Ser Gly Val Pro Phe Leu Gly Pro Tyr His Lys Glu Ser Ala Val Thr 130 135 140 Ala Phe Ser Glu Gly Ser Val Ile Ala Tyr Tyr Trp Ser Glu Phe Ser 145 150 155 160 Ile Pro Gln His Leu Val Glu Glu Ala Glu Arg Val Met Ala Glu Glu 165 170 175 Arg Val Val Met Leu Pro Pro Arg Ala Arg Ser Leu Lys Ser Phe Val 180 185 190 Val Thr Ser Val Val Ala Phe Pro Thr Asp Ser Lys Thr Val Gln Arg 195 200 205 Thr Gln Asp Asn Ser Cys Ser Phe Gly Leu His Ala Arg Gly Val Glu 210 215 220 Leu Met Arg Phe Thr Thr Pro Gly Phe Pro Asp Ser Pro Tyr Pro Ala 225 230 235 240 His Ala Arg Cys Gln Trp Ala Leu Arg Gly Asp Ala Asp Ser Val Leu 245 250 255 Ser Leu Thr Phe Arg Ser Phe Asp Leu Ala Ser Cys Asp Glu Arg Gly 260 265 270 Ser Asp Leu Val Thr Val Tyr Asn Thr Leu Ser Pro Met Glu Pro His 275 280 285 Ala Leu Val Gln Leu Cys Gly Thr Tyr Pro Pro Ser Tyr Asn Leu Thr 290 295 300 Phe His Ser Ser Gln Asn Val Leu Leu Ile Thr Leu Ile Thr Asn Thr 305 310 315 320 Glu Arg Arg His Pro Gly Phe Glu Ala Thr Phe Phe Gln Leu Pro Arg 325 330 335 Met Ser Ser Cys Gly Gly Arg Leu Arg Lys Ala Gln Gly Thr Phe Asn 340 345 350 Ser Pro Tyr Tyr Pro Gly His Tyr Pro Pro Asn Ile Asp Cys Thr Trp 355 360 365 Asn Ile Glu Val Pro Asn Asn Gln His Val Lys Val Arg Phe Lys Phe 370 375 380 Phe Tyr Leu Leu Glu Pro Gly Val Pro Ala Gly Thr Cys Pro Lys Asp 385 390 395 400 Tyr Val Glu Ile Asn Gly Glu Lys Tyr Cys Gly Glu Arg Ser Gln Phe 405 410 415 Val Val Thr Ser Asn Ser Asn Lys Ile Thr Val Arg Phe His Ser Asp 420 425 430 Gln Ser Tyr Thr Asp Thr Gly Phe Leu Ala Glu Tyr Leu Ser Tyr Asp 435 440 445 Ser Ser Asp Pro Cys Pro Gly Gln Phe Thr Cys Arg Thr Gly Arg Cys 450 455 460 Ile Arg Lys Glu Leu Arg Cys Asp Gly Trp Ala Asp Cys Thr Asp His 465 470 475 480 Ser Asp Glu Leu Asn Cys Ser Cys Asp Ala Gly His Gln Phe Thr Cys 485 490 495 Lys Asn Lys Phe Cys Lys Pro Leu Phe Trp Val Cys Asp Ser Val Asn 500 505 510 Asp Cys Gly Asp Asn Ser Asp Glu Gln Gly Cys Ser Cys Pro Ala Gln 515 520 525 Thr Phe Arg Cys Ser Asn Gly Lys Cys Leu Ser Lys Ser Gln Gln Cys 530 535 540 Asn Gly Lys Asp Asp Cys Gly Asp Gly Ser Asp Glu Ala Ser Cys Pro 545 550 555 560 Lys Val Asn Val Val Thr Cys Thr Lys His Thr Tyr Arg Cys Leu Asn 565 570 575 Gly Leu Cys Leu Ser Lys Gly Asn Pro Glu Cys Asp Gly Lys Glu Asp 580 585 590 Cys Ser Asp Gly Ser Asp Glu Lys Asp Cys Asp Cys Gly Leu Arg Ser 595 600 605 Phe Thr Arg Gln Ala Arg Val Val Gly Gly Thr Asp Ala Asp Glu Gly 610 615 620 Glu Trp Trp Gln Val Ser Leu His Ala Leu Gly Gln Gly His Ile Cys 625 630 635 640 Gly Ala Ser Leu Ile Ser Pro Asn Trp Leu Val Ser Ala Ala His Cys 645 650 655 Tyr Ile Asp Asp Arg Gly Phe Arg Tyr Ser Asp Pro Thr Gln Trp Thr 660 665 670 Ala Phe Leu Gly Leu His Asp Gln Ser Gln Arg Ser Ala Pro Gly Val 675 680 685 Gln Glu Arg Arg Leu Lys Arg Ile Ile Ser His Pro Phe Phe Asn Asp 690 695 700 Phe Thr Phe Asp Tyr Asp Ile Ala Leu Leu Glu Leu Glu Lys Pro Ala 705 710 715 720 Glu Tyr Ser Ser Met Val Arg Pro Ile Cys Leu Pro Asp Ala Ser His 725 730 735 Val Phe Pro Ala Gly Lys Ala Ile Trp Val Thr Gly Trp Gly His Thr 740 745 750 Gln Tyr Gly Gly Thr Gly Ala Leu Ile Leu Gln Lys Gly Glu Ile Arg 755 760 765 Val Ile Asn Gln Thr Thr Cys Glu Asn Leu Leu Pro Gln Gln Ile Thr 770 775 780 Pro Arg Met Met Cys Val Gly Phe Leu Ser Gly Gly Val Asp Ser Cys 785 790 795 800 Gln Gly Asp Ser Gly Gly Pro Leu Ser Ser Val Glu Ala Asp Gly Arg 805 810 815 Ile Phe Gln Ala Gly Val Val Ser Trp Gly Asp Gly Cys Ala Gln Arg 820 825 830 Asn Lys Pro Gly Val Tyr Thr Arg Leu Pro Leu Phe Arg Asp Trp Ile 835 840 845 Lys Glu Asn Thr Gly Val His His His His His 850 855 860 <210> 101 <211> 849 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" "Polypeptide" <400> 101 Met Asp Lys Glu Asn Ser Asp Val Ser Ala Ala Pro Ala Asp Leu Lys 1 5 10 15 Ile Ser Asn Ile Ser Val Gln Val Val Ser Ala Gln Lys Lys Leu Pro 20 25 30 Val Arg Arg Pro Pro Leu Pro Gly Arg Arg Leu Pro Leu Pro Gly Arg 35 40 45 Arg Pro Pro Gln Arg Pro Ile Gly Lys Ala Lys Pro Lys Lys Gln Ser 50 55 60 Light Light Light Val Pro Phe Trp Asn Val Gln Asn Light Ile Ile Leu Phe 65 70 75 80 Thr Val Phe Leu Phe Ile Leu Ala Val Ile Ala Trp Thr Leu Leu Trp 85 90 95 Leu Tyr Ile Ser Lys Thr Glu Ser Lys Asp Ala Phe Tyr Phe Ala Gly 100 105 110 Met Phe Arg Ile Thr Asn Ile Glu Phe Leu Pro Glu Tyr Arg Gln Lys 115 120 125 Glu Ser Arg Glu Phe Leu Ser Val Ser Arg Thr Val Gln Gln Val Ile 130 135 140 Asn Leu Val Tyr Thr Thr Ser Ala Phe Ser Lys Phe Tyr Glu Gln Ser 145 150 155 160 Val Val Ala Asp Val Ser Ser Asn Asn Lys Gly Gly Leu Leu Val His 165 170 175 Phe Trp Ile Val Phe Val Met Pro Arg Ala Lys Gly His Ile Phe Cys 180 185 190 Glu Asp Cys Val Ala Ala Ile Leu Lys Asp Ser Ile Gln Thr Ser Ile 195 200 205 Ile Asn Arg Thr Ser Val Gly Ser Leu Gln Gly Leu Ala Val Asp Met 210 215 220 Asp Ser Val Val Leu Asn Ala Gly Leu Arg Ser Asp Tyr Ser Ser Thr 225 230 235 240 Ile Gly Ser Asp Lys Gly Cys Ser Gln Tyr Phe Tyr Ala Glu His Leu 245 250 255 Ser Leu His Tyr Pro Leu Glu Ile Ser Ala Ala Ser Gly Arg Leu Met 260 265 270 Cys His Phe Lys Leu Val Ala Ile Val Gly Tyr Leu Ile Arg Leu Ser 275 280 285 Ile Lys Ser Ile Gln Ile Glu Ala Asp Asn Cys Val Thr Asp Ser Leu 290 295 300 Thr Ile Tyr Asp Ser Leu Leu Pro Ile Arg Ser Ser Ile Leu Tyr Arg 305 310 315 320 Ile Cys Glu Pro Thr Arg Thr Leu Met Ser Phe Val Ser Thr Asn Asn 325 330 335 Leu Met Leu Val Thr Phe Lys Ser Pro His Ile Arg Arg Leu Ser Gly 340 345 350 Ile Arg Ala Tyr Phe Glu Val Ile Pro Glu Gln Lys Cys Glu Asn Thr 355 360 365 Val Leu Val Lys Asp Ile Thr Gly Phe Glu Gly Lys Ile Ser Ser Pro 370 375 380 Tyr Tyr Pro Ser Tyr Tyr Pro Pro Lys Cys Lys Cys Thr Trp Lys Phe 385 390 395 400 Gln Thr Ser Leu Ser Thr Leu Gly Ile Ala Leu Lys Phe Tyr Asn Tyr 405 410 415 Ser Ile Thr Lys Lys Ser Met Lys Gly Cys Glu His Gly Trp Trp Glu 420 425 430 Ile Asn Glu His Met Tyr Cys Gly Ser Tyr Met Asp His Gln Thr Ile 435 440 445 Phe Arg Val Pro Ser Pro Leu Val His Ile Gln Leu Gln Cys Ser Ser 450 455 460 Arg Leu Ser Asp Lys Pro Leu Leu Ala Glu Tyr Gly Ser Tyr Asn Ile 465 470 475 480 Ser Gln Pro Cys Pro Val Gly Ser Phe Arg Cys Ser Ser Gly Leu Cys 485 490 495 Val Pro Gln Ala Gln Arg Cys Asp Gly Val Asn Asp Cys Phe Asp Glu 500 505 510 Ser Asp Glu Leu Phe Cys Val Ser Pro Gln Pro Ala Cys Asn Thr Ser 515 520 525 Ser Phe Arg Gln His Gly Pro Leu Ile Cys Asp Gly Phe Arg Asp Cys 530 535 540 Glu Asn Gly Arg Asp Glu Gln Asn Cys Thr Gln Ser Ile Pro Cys Asn 545 550 555 560 Asn Arg Thr Phe Lys Cys Gly Asn Asp Ile Cys Phe Arg Lys Gln Asn 565 570 575 Ala Lys Cys Asp Gly Thr Val Asp Cys Pro Asp Gly Ser Asp Glu Glu 580 585 590 Gly Cys Thr Cys Ser Arg Ser Ser Ser Ala Leu His Arg Ile Ile Gly 595 600 605 Gly Thr Asp Thr Leu Glu Gly Gly Trp Pro Trp Gln Val Ser Leu His 610 615 620 Phe Val Gly Ser Ala Tyr Cys Gly Ala Ser Val Ile Ser Arg Glu Trp 625 630 635 640 Leu Leu Ser Ala Ala His Cys Phe His Gly Asn Arg Leu Ser Asp Pro 645 650 655 Thr Pro Trp Thr Ala His Leu Gly Met Tyr Val Gln Gly Asn Ala Lys 660 665 670 Phe Val Ser Pro Val Arg Arg Ile Val Val His Glu Tyr Tyr Asn Ser 675 680 685 Gln Thr Phe Asp Tyr Asp Ile Ala Leu Leu Gln Leu Ser Ile Ala Trp 690 695 700 Pro Glu Thr Leu Lys Gln Leu Ile Gln Pro Ile Cys Ile Pro Pro Thr 705 710 715 720 Gly Gln Arg Val Arg Ser Gly Glu Lys Cys Trp Val Thr Gly Trp Gly 725 730 735 Arg Arg His Glu Ala Asp Asn Lys Gly Ser Leu Val Leu Gln Gln Ala 740 745 750 Glu Val Glu Leu Ile Asp Gln Thr Leu Cys Val Ser Thr Tyr Gly Ile 755 760 765 Ile Thr Ser Arg Met Leu Cys Ala Gly Ile Met Ser Gly Lys Arg Asp 770 775 780 Ala Cys Lys Gly Asp Ser Gly Gly Pro Leu Ser Cys Arg Arg Lys Ser 785 790 795 800 Asp Gly Lys Trp Ile Leu Thr Gly Ile Val Ser Trp Gly His Gly Ser 805 810 815 Gly Arg Pro Asn Phe Pro Gly Val Tyr Thr Arg Val Ser Asn Phe Val 820 825 830 Pro Trp Ile His Lys Tyr Val Pro Ser Leu Leu His His His His 835 840 845 His <210> 102 <211> 783 <212> PRT <213> Artificial sequence <220> <221> source <223> / Note="Description of artificial sequence: synthesis" "Polypeptide" <400> 102 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Arg Gly Ala Asp Ile Gly Tyr Lys Ala Glu Val Met 20 25 30 Val Ser Gln Val Tyr Ser Gly Ser Leu Arg Val Leu Asn Arg His Phe 35 40 45 Ser Gln Asp Leu Thr Arg Arg Glu Ser Ser Ala Phe Arg Ser Glu Thr 50 55 60 Ala Lys Ala Gln Lys Met Leu Lys Glu Leu Ile Thr Ser Thr Arg Leu 65 70 75 80 Gly Thr Tyr Tyr Asn Ser Ser Ser Val Tyr Ser Phe Gly Glu Gly Pro 85 90 95 Leu Thr Cys Phe Phe Trp Phe Ile Leu Gln Ile Pro Glu His Arg Arg 100 105 110 Leu Met Leu Ser Pro Glu Val Val Gln Ala Leu Leu Val Glu Glu Leu 115 120 125 Leu Ser Thr Val Asn Ser Ser Ala Ala Val Pro Tyr Arg Ala Glu Tyr 130 135 140 Glu Val Asp Pro Glu Gly Leu Val Ile Leu Glu Ala Ser Val Lys Asp 145 150 155 160 Ile Ala Ala Leu Asn Ser Thr Leu Gly Cys Tyr Arg Tyr Ser Tyr Val 165 170 175 Gly Gln Gly Gln Val Leu Arg Leu Lys Gly Pro Asp His Leu Ala Ser 180 185 190 Ser Cys Leu Trp His Leu Gln Gly Pro Lys Asp Leu Met Leu Lys Leu 195 200 205 Arg Leu Glu Trp Thr Leu Ala Glu Cys Arg Asp Arg Leu Ala Met Tyr 210 215 220 Asp Val Ala Gly Pro Leu Glu Lys Arg Leu Ile Thr Ser Val Tyr Gly 225 230 235 240 Cys Ser Arg Gln Glu Pro Val Val Glu Val Leu Ala Ser Gly Ala Ile 245 250 255 Met Ala Val Val Trp Lys Lys Gly Leu His Ser Tyr Tyr Asp Pro Phe 260 265 270 Val Leu Ser Val Gln Pro Val Val Phe Gln Ala Cys Glu Val Asn Leu 275 280 285 Thr Leu Asp Asn Arg Leu Asp Ser Gln Gly Val Leu Ser Thr Pro Tyr 290 295 300 Phe Pro Ser Tyr Tyr Ser Pro Gln Thr His Cys Ser Trp His Leu Thr 305 310 315 320 Val Pro Ser Leu Asp Tyr Gly Leu Ala Leu Trp Phe Asp Ala Tyr Ala 325 330 335 Leu Arg Arg Gln Lys Tyr Asp Leu Pro Cys Thr Gln Gly Gln Trp Thr 340 345 350 Ile Gln Asn Arg Arg Leu Cys Gly Leu Arg Ile Leu Gln Pro Tyr Ala 355 360 365 Glu Arg Ile Pro Val Val Ala Thr Ala Gly Ile Thr Ile Asn Phe Thr 370 375 380 Ser Gln Ile Ser Leu Thr Gly Pro Gly Val Arg Val His Tyr Gly Leu 385 390 395 400 Tyr Asn Gln Ser Asp Pro Cys Pro Gly Glu Phe Leu Cys Ser Val Asn 405 410 415 Gly Leu Cys Val Pro Ala Cys Asp Gly Val Lys Asp Cys Pro Asn Gly 420 425 430 Leu Asp Glu Arg Asn Cys Val Cys Arg Ala Thr Phe Gln Cys Lys Glu 435 440 445 Asp Ser Thr Cys Ile Ser Leu Pro Lys Val Cys Asp Gly Gln Pro Asp 450 455 460 Cys Leu Asn Gly Ser Asp Glu Glu Gln Cys Gln Glu Gly Val Pro Cys 465 470 475 480 Gly Thr Phe Thr Phe Gln Cys Glu Asp Arg Ser Cys Val Lys Lys Pro 485 490 495 Asn Pro Gln Cys Asp Gly Arg Pro Asp Cys Arg Asp Gly Ser Asp Glu 500 505 510 Glu His Cys Asp Cys Gly Leu Gln Gly Pro Ser Ser Arg Ile Val Gly 515 520 525 Gly Ala Val Ser Ser Glu Gly Glu Trp Pro Trp Gln Ala Ser Leu Gln 530 535 540 Val Arg Gly Arg His Ile Cys Gly Gly Ala Leu Ile Ala Asp Arg Trp 545 550 555 560 Val Ile Thr Ala Ala His Cys Phe Gln Glu Asp Ser Met Ala Ser Thr 565 570 575 Val Leu Trp Thr Val Phe Leu Gly Lys Val Trp Gln Asn Ser Arg Trp 580 585 590 Pro Gly Glu Val Ser Phe Lys Val Ser Arg Leu Leu Leu His Pro Tyr 595 600 605 His Glu Glu Asp Ser His Asp Tyr Asp Val Ala Leu Leu Gln Leu Asp 610 615 620 His Pro Val Val Arg Ser Ala Ala Val Arg Pro Val Cys Leu Pro Ala 625 630 635 640 Arg Ser His Phe Phe Glu Pro Gly Leu His Cys Trp Ile Thr Gly Trp 645 650 655 Gly Ala Leu Arg Glu Gly Gly Pro Ile Ser Asn Ala Leu Gln Lys Val 660 665 670 Asp Val Gln Leu Ile Pro Gln Asp Leu Cys Ser Glu Val Tyr Arg Tyr 675 680 685 Gln Val Thr Pro Arg Met Leu Cys Ala Gly Tyr Arg Lys Gly Lys Lys 690 695 700 Asp Ala Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Lys Ala Leu 705 710 715 720 Ser Gly Arg Trp Phe Leu Ala Gly Leu Val Ser Trp Gly Leu Gly Cys 725 730 735 Gly Arg Pro Asn Tyr Phe Gly Val Tyr Thr Arg Ile Thr Gly Val Ile 740 745 750 Ser Trp Ile Gln Gln Val Val Thr Gly Gly Gly Gly Ser Gly Gly Gly 755 760 765 Gly Ser Gly Gly Gly Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys 770 775 780

Claims

1. An anti-TMPRSS6 antibody, which is a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human TMPRSS6 and increases the activity of the hepcidin promoter, wherein the antibody is capable of binding to human TMPRSS6 on the cell surface expressing human TMPRSS6 and includes one of the following: i. (a) a heavy chain polypeptide comprising HC CDR1 having the sequence GFNIEDYY as described in SEQ ID NO: 22, HC CDR2 having the sequence IDPEDGET as described in SEQ ID NO: 23, and HC CDR3 having the sequence ARSIYLDPMDY as described in SEQ ID NO: 24; and (b) a light chain polypeptide comprising LC CDR1 having the sequence QDVTTA as described in SEQ ID NO: 27 or SEQ ID NO: 57, LC CDR2 having the sequence WAT as described in SEQ ID NO: 58, and LC CDR3 having the sequence QQHYSTPYT as described in SEQ ID NO: 29; or ii. (a) a heavy chain polypeptide comprising HC CDR1 having the sequence GFNIEDYY as set forth in SEQ ID NO: 52, HC CDR2 having the sequence IDPEDAET as set forth in SEQ ID NO: 53, and HC CDR3 having the sequence ARSIYLDPMDY as set forth in SEQ ID NO: 54; and (b) a light chain polypeptide comprising LC CDR1 having the sequence QDVTTA as set forth in SEQ ID NO: 57, LC CDR2 having the sequence WAT as set forth in SEQ ID NO: 58, and LC CDR3 having the sequence QQHYSTPYT as set forth in SEQ ID NO:

59.

2. The anti-TMPRSS6 antibody according to claim 1, wherein the anti-TMPRSS6 antibody is capable of at least one of the following: inhibiting TMPRSS6-induced hepcidin expression; increasing hepcidin expression; and inhibiting TMPRSS6-induced hepcidin expression induced by the BMP / SMAD pathway.

3. The anti-TMPRSS6 antibody according to claim 1, wherein the antibody exhibits cross-reactivity with at least one non-human TMPRSS6.

4. The anti-TMPRSS6 antibody according to claim 3, wherein the non-human TMPRSS6 is at least one of mouse TMPRSS6 or cynomolgus monkey TMPRSS6.

5. The anti-TMPRSS6 antibody according to claim 1, wherein the anti-TMPRSS6 antibody specifically binds to human TMPRSS6 (interstitial protease-2) and does not exhibit detectable binding to interstitial protease homologs selected from at least one of human interstitial protease-1 (ST14) or human interstitial protease-3 (TMPRSS7).

6. The anti-TMPRSS6 antibody according to claim 1, wherein the antibody is a chimeric antibody.

7. The anti-TMPRSS6 antibody according to claim 1, wherein the antibody is a humanized antibody.

8. The anti-TMPRSS6 antibody according to claim 1, wherein the antibody is an antigen-binding fragment.

9. The anti-TMPRSS6 antibody according to claim 1, comprising at least one polypeptide having an amino acid sequence selected from the following: SEQ ID NO: 21; SEQ ID NO: 26; SEQ ID NO: 51; SEQ ID NO: 56; SEQ ID NO: 69; SEQ ID NO: 71; SEQ ID NO: 81 or SEQ ID NO:

83.

10. The anti-TMPRSS6 antibody according to claim 1, wherein the antibody comprises: (a) a heavy chain (HC) polypeptide, wherein the variable region comprises an amino acid sequence selected from the amino acid sequences of SEQ ID NO: 21 and SEQ ID NO: 51; and (b) a light chain (LC) polypeptide, wherein the variable region comprises an amino acid sequence selected from the amino acid sequences of SEQ ID NO: 26 and SEQ ID NO:

56.

11. Use of the anti-TMPRSS6 antibody according to claim 1 in the manufacture of a medicament for treating iron overload syndrome, wherein the iron overload syndrome is β-thalassemia, and wherein the medicament increases the activity of the hepcidin promoter.

12. A pharmaceutical composition comprising the anti-TMPRSS6 antibody according to claim 1 and a suitable carrier and / or excipient.

13. An isolated nucleic acid molecule encoding the anti-TMPRSS6 antibody according to claim 1.

14. The isolated nucleic acid molecule according to claim 13, comprising a heavy chain (HC) nucleotide sequence selected from the following: a nucleotide sequence comprising SEQ ID NO: 25 or a nucleotide sequence comprising SEQ ID NO:

55.

15. The isolated nucleic acid molecule according to claim 13, comprising a light chain (LC) nucleotide sequence selected from the group consisting of a nucleotide sequence comprising SEQ ID NO: 30 or a nucleotide sequence comprising SEQ ID NO:

60.

16. A vector comprising the nucleic acid molecule according to claim 13.

17. A host cell comprising the vector according to claim 16.

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