Treatment of physiologic iron overload

Abstract

Antibodies against the protease 2 (MTP-2) are provided. Inhibition of MTP-2 reduces dietary iron uptake and reduces release of iron from cellular stores in the body. Inhibitors of MTP-2, such as antibodies against the serine protease domain, are useful in the treatment of iron overload, which is characteristic of diseases such as beta-thalassemia and would otherwise result in toxic accumulation of iron. Combinations of MTP-2 inhibitors with activin receptor ligand traps or with erythropoietin provide additional therapeutic effects.

Description

TECHNICAL FIELD

[0001] The present invention relates to a medicament for reducing iron overload in patients suffering from conditions such as beta-thalassemia and myelodysplastic syndrome (MDS). BACKGROUND

[0002] Iron is essential for erythropoiesis, the production of red blood cells that transport oxygen from the lungs to other tissues of the body. However, excess iron is toxic because it can generate reactive oxygen species, so its absorption from the duodenum must be tightly regulated. The liver peptide hormone hepcidin plays an important role in regulating iron absorption to meet the body's iron demand. Hepcidin negatively regulates cellular iron export by promoting the degradation of ferroportin, the only known iron exporter. Ferroportin is expressed on the basolateral side of cells such as macrophages and duodenal enterocytes that serve as major storage compartments for iron. Thus, hepcidin's negative regulation of ferroportin limits duodenal iron absorption and release of iron from iron storage cells such as macrophages. It has been determined that activation of the BMP-SMAD signaling pathway in hepatocytes by secretion of BMP ligands, primarily BMP6, stimulates hepcidin expression. Matriptase-2 (MTP-2), a type II transmembrane trypsin-like serine protease encoded by the gene Tmprss6, is expressed by hepatocytes and inhibits BMP-SMAD signaling by potentially cleaving BMP co-receptors such as HJV, thereby decreasing hepcidin expression and causing increased uptake of dietary iron and release of iron from cellular storage. Expression of MTP-2 is induced by BMP6 and excess iron, which is required for hepcidin expression (Meynard et al., Blood 118:747-756 2011). Thus, while BMP6 stimulates hepcidin expression, it also increases the expression of a negative regulator, maintaining a negative feedback mechanism to prevent iron dysregulation (Wahedi et al., J Biol Chem 292:18354-18371 2017). Figure 1 .

[0003] Iron overload, manifested as higher than normal transferrin iron saturation in the blood, contributes to the pathogenesis of many genetic diseases and other conditions including beta- thalassemia, myelodysplastic syndrome (MDS), Blackfan Diamond anemia, sickle cell disease, polycythemia vera, and hemochromatosis.

[0004] Beta-thalassemia is a genetic hemoglobinopathy caused by genetic defects in the beta-globin gene. In adults, hemoglobin is normally composed of four polypeptide (globin) chains - two alpha-globin subunits and two beta-globin subunits - each globin subunit carrying a heme group with a central iron that reversibly binds oxygen. In beta-thalassemia, the defective production of hemoglobin leads to ineffective erythropoiesis and thus to anemia (lack of oxygen-carrying red blood cells). The overproduction of erythropoietin (epo), which is upregulated in response to anemia, and / or the elevated levels of erythroferrone, a hormone produced by abnormal expansion and ineffective erythropoiesis, have an inhibitory effect on the iron regulator hepcidin, resulting in increased absorption of iron from the gut and release of iron from internal stores, leading to iron overload. Interestingly, it is not anemia itself but iron overload that is believed to reduce life expectancy in beta-thalassemia. The increased availability of iron leads to increased transferrin saturation and heme production, which, together with a compensatory increase in alpha-globin expression, results in increased formation of high-iron hemochromogens and active oxygen stress and increased apoptosis of erythroid progenitors. This highly stimulated but ineffective erythropoiesis leads to a large number of mature red blood cells that do not survive, thus leading to splenomegaly or splenomegaly, which is also a feature of the disease.

[0005] There are three types of beta-thalassemia, classified according to the degree of reduction in beta-chain synthesis. Homozygous or severe beta-thalassemia is the most severe form of congenital hemolytic anemia, characterized by a lack or severe inhibition of functional beta-chain synthesis. Patients require very frequent transfusions ("transfusion-dependent beta-thalassemia"). On the other hand, intermediate beta-thalassemia patients do not require regular transfusions and are genetically heterozygous ("non-transfusion-dependent beta-thalassemia"). The third form of beta-thalassemia is called mild beta-thalassemia, a mild, asymptomatic condition in which beta-chain synthesis is only moderately inhibited.

[0006] One adverse effect of transfusions in transfusion-dependent patients is that each transfusion contains at least 200 mg of iron, thus exacerbating the problem of iron overload and toxic tissue damage caused by non-transferrin-bound iron, which requires iron chelation therapy. Iron chelators complex with non-transferrin-bound iron in the circulation and can shift the balance and release iron from tissues to avoid tissue damage. However, chelators do not lower transferrin saturation and thus do not avoid increased formation of high-iron hemochromogens and increased apoptosis of cells. Patients remain dependent on transfusions. In addition, iron chelators can have side effects such as renal failure, toxic neutropenia, and diarrhea. Although transfusions and iron chelation have improved the prognosis of transfusion-dependent patients, the iron overload experienced by some patients today still represents an unmet clinical need.

[0007] Similarly, there is an unmet clinical need for treating patients diagnosed with other iron overload anemias and related disorders. Myelodysplastic syndromes (MDS) are a group of clonal stem cell disorders characterized by ineffective and dysplastic hematopoiesis leading to anemia and cytopenias, and a different propensity to develop acute myeloid leukemia (AML). Several forms of MDS are associated with anemia and toxic iron deposition in erythroid precursors, including MDS with 5q- chromosomal deletion (5q-MDS) and refractory anemia with ring sideroblasts (RARS). These forms of MDS are often associated with decreased levels of hepcidin. Typically, MDS can be managed with regular transfusions, but this can lead to secondary iron overload and decrease overall survival, as in beta-thalassemia. Thus, MDS patients are often treated with iron chelators when iron overload reaches a certain threshold.

[0008] Another iron overload disease is hereditary hemochromatosis. It is the most common genetic disease in Caucasians, characterized by genetic mutations that cause excess iron absorption and accumulation due to hepcidin deficiency or insensitivity. Type 1 hemochromatosis is caused by mutations in the HFE gene. Type 2 hemochromatosis is caused by mutations in the HJV or HAMP gene. Type 3 hemochromatosis is caused by mutations in the TFR2 gene. Type 4 hemochromatosis is caused by mutations in the SLC40A1 gene. Symptoms of the disease include joint pain, abdominal pain, fatigue, and weakness. If left untreated, the disease can lead to cirrhosis of the liver, liver cancer, heart disease and / or failure, and diabetes. Current treatment is venesection.

[0009] Rare forms of anemia have also been shown to benefit from iron-reducing therapies in animal models. These include Blackfan Diamond anemia and sickle cell anemia. Iron deposition within the liver, primarily in parenchymal cells, is also thought to contribute to oxidative stress and fibrosis in diseases such as hemochromatosis and hepatitis C infection, as well as iron-loading anemias. Regulation of iron metabolism is also thought to be important in the development of liver fibrosis and cirrhosis, nonalcoholic fatty liver disease (NAFDL), and nonalcoholic steatohepatitis (NASH). Liver fibrosis often transforms into cirrhosis, with loss of liver function and progression to liver cancer.

[0010] New medical treatments are being sought for patients with conditions such as those described above. While transfusions and iron chelation can improve overall survival, these treatments do not address the underlying disease pathology and have undesirable side effects, as noted.

[0011] One branch of the research involves the biological study of iron overload anemia, which uses mice with inherited β-thalassemia as a model of the human disease. Hbbth3 / + mice exhibit similar characteristics to human intermediate β-thalassemia, including Hb levels between 7 and 9 g / dL, abnormal red blood cell morphology, increased reticulocyte count,

[0012] ineffective and extramedullary erythropoiesis, hepatosplenomegaly, and liver and spleen iron overload, a complex phenotype that worsens with age (Franceschi et al., Haematologica 91 : 1336-134 2006).

[0013] Knocking out or reducing expression of Tmprss6 (MTP2) has been shown to increase hepcidin expression, correcting iron overload, splenomegaly, and anemia in Hbbth3 / + mice (Guo et al., Journal of Clinical Investigation 123: 1531-1541 2013; Nai et al., Blood 119: 5021-5029 2012). It has been shown that reducing gene expression of Tmprss6 by using Tmprss6 siRNA formulated in lipid nanoparticles (LNP) in the Hbbth3 / + mouse model of β-thalassemia induces hepcidin and reduces tissue and serum iron levels. In addition, LNP-Tmprss6 siRNA treatment of Hbbth3 / + animals improves red blood cell survival and erythropoiesis and thus significantly reduces anemia (Schmidt et al., Blood 121(7): 1200-1208 2013).

[0014] Guo et al. demonstrated that Hbbth3 / + mice showed reduced insoluble membrane-bound globin formation, ROS and apoptosis, and anemia after treatment with an antisense oligonucleotide against Tmprss6 (Guo et al., 2013, supra). These animals also exhibited lower levels of erythropoietin, a significant improvement in ineffective erythropoiesis and splenomegaly, and an increase in total hemoglobin levels.

[0015] Consistent with these studies, knocking out Tmprss6 in the genetic background of thalassemic mice significantly reduced iron overload and increased hemoglobin levels compared to Tmprss6+ thalassemic mice.

[0016] Gene therapy represents a possible approach to addressing the underlying disease pathology in patients with iron overload disorders. In June 2019, the gene therapy “Zynteglo” received conditional marketing authorization from the EMA for the treatment of transfusion-dependent beta-thalassemia in patients aged 12 years and older who do not have other treatment options. The gene therapy adds a corrective beta-globin gene to the patient’s bone marrow stem cells ex vivo, which are then re-implanted into the patient. Despite the potential curative effect, this is a highly invasive and very expensive procedure. As of November 2019, the approval of Zynteglo was delayed due to production problems, and it has not yet been approved for the most severe type of beta-thalassemia (beta0 / beta0 genotype) because more than half of such patients treated experimentally with Zynteglo had to resume blood transfusions.

[0017] Also in clinical studies are fusion proteins containing the extracellular domain of the activin type II receptor linked to the Fc portion of human IgGl. These ligand traps act on the transforming growth factor-beta (TGF ) superfamily to increase late-stage erythropoiesis. Sotatercept is an activin type IIA receptor IgG-Fc fusion protein, and luspatercept is an activin type IIB receptor IgG-Fc fusion protein. These proteins have been shown to significantly reduce the need for red blood cell transfusions in iron overload anemia, but so far they have not been proven sufficient to achieve transfusion independence (Piga et al., Blood 133: 1279-1289 2019).

[0018] Based primarily on data provided by the phase III BELIEVE trial, luspatercept (Reblozyl) received FDA approval on November 8, 2019 for the treatment of anemia in adults with beta thalassemia who require regular red blood cell transfusions. The primary clinical endpoint of the study was the proportion of patients achieving a reduction of at least 33% in transfusion burden from baseline, a reduction of at least 2 units from week 13 to week 24. This was achieved by only 21.4% of patients, and only 7.6% to 10.3% of patients achieved a reduction in transfusions of more than 50% over the same time frame. Thus, there remains a continuing medical need for treatment of beta thalassemia.

[0019] Perhaps the simplest concept for increasing hepcidin levels through therapeutic intervention is the therapeutic use of hepcidin, hepcidin derivatives or analogs themselves (Casu et al., Blood 128:265-276 2016; Casu, Nemeth & Rivella, Blood 131 : 1790-1794 2018; Preza et al., Journal of Clinical Investigation 121 :4880-4888, 2011). Despite being a simple therapeutic concept, no such therapy has yet succeeded in meeting the standards for regulatory approval. At least two of these approaches have been terminated following recent clinical trials (LJPC-401 in a phase II trial of beta-thalassemia by La Jolla Pharmaceuticals and M-021 following a phase I trial by Merganser). Moreover, there remains a medical need to develop effective new therapies. SUMMARY

[0020] The present invention relates to binding polypeptides, such as antibodies, that bind to and inhibit MTP-2. MTP-2-inhibiting binding polypeptides are useful for reducing iron overload in patients, including patients with beta-thalassemia, MSD and other iron overload anemias, and other conditions described herein. Aspects of the invention relate to binding polypeptides, their use in the manufacture of medicaments and in methods of treating patients, methods of producing binding polypeptides, nucleic acids encoding the binding polypeptides, and pharmaceutical formulations containing the binding polypeptides.

[0021] In a first aspect, the invention provides a binding polypeptide that binds to MTP-2 and inhibits the enzymatic activity of the MTP-2. The binding polypeptide may, for example, bind to the serine protease catalytic domain of MTP-2.

[0022] A binding polypeptide according to the invention can be an antibody (e.g., an IgG) or a non-antibody molecule, such as an alternative polypeptide scaffold comprising an engineered binding loop. We describe antibodies and other binders comprising binding loops directed to MTP-2 (e.g., the MTP-2 serine protease catalytic domain) that inhibit the enzymatic activity of MTP-2.

[0023] Embodiments include antibodies referred to herein as NORI-001, NORI-002, NORI-003, NORI-004, NORI-005, NORI-006, NORI-007, NORI-008, NORI-009, NORI-010, NORI-011, NORI-012, NORI-013, NORI-014, NORI-015, NORI-016, NORI-017, NORI-018, NORI-019, NORI-020, NORI-021, NORI-022, NORI-023, NORI-024, NORI-025, NORI-026, NORI-027, NORI-028, NORI-029, NORI-030, NORI-031, NORI-032, and NORI-033 ("NORI-001 to NORI-033"). These represent different selections of antibody heavy and light chain sequences. These antibodies were all shown to bind MTP-2 and inhibit the catalytic activity of said MTP-2. Selected example antibodies also showed successful reduction of hepcidin expression in vivo, which represents the modulation of a key biological process in physiologic iron overload, and suggest the potential of inhibitory anti-MTP-2 binding polypeptides to treat blood disorders and other conditions associated with iron overload.

[0024] The binding polypeptide according to the present application can be an antibody comprising a VH domain comprising a set of heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3, and a VL domain comprising a set of light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3.

[0025] The antibody can comprise a HCDR1, HCDR2, and / or HCDR3 that is a HCDR1, HCDR2, or HCDR3 of any one of NORI-001 to NORI-033; and / or the antibody can comprise a LCDR1, LCDR2, or LCDR3 that is a LCDR1, LCDR2, or LCDR3 of any one of NORI-001 to NORI-033. For example, the antibody can comprise the HCDR3 of NORI-001, NORI-002, NORI-003, NORI-004, NORI-005, NORI-006, NORI-007, NORI-008, NORI-009, NORI-010, NORI-011, NORI-012, NORI-013, NORI-014, NORI-015, NORI-016, NORI-017, NORI-018, NORI-019, NORI-020, NORI-021, NORI-022, NORI-023, NORI-024, NORI-025, NORI-026, NORI-027, NORI-028, NORI-029, NORI-030, NORI-031, NORI-032, or NORI-033.

[0026] The antibody can comprise a set of HCDRs that is a set of HCDRs of the VH domain of any one of NORI-001 to NORI-033; and / or the antibody can comprise a set of LCDRs that is a set of LCDRs of the VL domain of any one of NORI-001 to NORI-033. For example, the antibody can comprise the HCDRs and LCDRs of NORI-001, NORI-002, NORI-003, NORI-004, NORI-005, NORI-006, NORI-007, NORI-008, NORI-009, NORI-010, NORI-011, NORI-012, NORI-013, NORI-014, NORI-015, NORI-016, NORI-017, NORI-018, NORI-019, NORI-020, NORI-021, NORI-022, NORI-023, NORI-024, NORI-025, NORI-026, NORI-027, NORI-028, NORI-029, NORI-030, NORI-031, NORI-032, or NORI-033.

[0027] The binding polypeptide can comprise a VH domain having at least 90% amino acid sequence identity to the VH domain of any one of NORI-001 to NORI-033 and / or it can comprise a VL domain having at least 90% amino acid sequence identity to the VL domain of any one of NORI-001 to NORI-033. For example, the binding polypeptide can comprise the VH and VL domains of NORI-001, NORI-002, NORI-003, NORI-004, NORI-005, NORI-006, NORI-007, NORI-008, NORI-009, NORI-010, NORI-011, NORI-012, NORI-013, NORI-014, NORI-015, NORI-016, NORI-017, NORI-018, NORI-019, NORI-020, NORI-021, NORI-022, NORI-023, NORI-024, NORI-025, NORI-026, NORI-027, NORI-028, NORI-029, NORI-030, NORI-031, NORI-032, or NORI-033, or a variant VH and / or VL domain sharing 90% sequence identity thereto.

[0028] In a first configuration, the antibody comprises

[0029] a VH domain comprising the NORI-003 HCDRs and having at least 90% sequence identity to the VH domain of NORI-003, and

[0030] a VL domain comprising the NORI-003 LCDRs and having at least 90% sequence identity to the VL domain of NORI-003.

[0031] The antibody can comprise the NORI-003 VH domain and the NORI-003 VL domain. Optionally, the antibody is an IgG comprising the NORI-003 heavy chain and the NORI-003 light chain.

[0032] In a second configuration, the antibody comprises

[0033] a VH domain comprising the NORI-006 HCDRs and having at least 90% sequence identity to the VH domain of NORI-006, and

[0034] a VL domain comprising the NORI-006 LCDRs and having at least 90% sequence identity to the VL domain of NORI-006.

[0035] The antibody can comprise a NORI-006 VH domain and a NORI-006 VL domain. Optionally, the antibody is an IgG comprising a NORI-006 heavy chain and a NORI-006 light chain.

[0036] In a third configuration, the antibody comprises

[0037] a VH domain comprising NORI-008 HCDRs and having at least 90% sequence identity to the VH domain of NORI-008, and

[0038] a VL domain comprising NORI-008 LCDRs and having at least 90% sequence identity to the VL domain of NORI-008.

[0039] The antibody can comprise a NORI-008 VH domain and a NORI-008 VL domain. Optionally, the antibody is an IgG comprising a NORI-008 heavy chain and a NORI-008 light chain.

[0040] In a fourth configuration, the antibody comprises

[0041] a VH domain comprising NORI-011 HCDRs and having at least 90% sequence identity to the VH domain of NORI-011, and

[0042] a VL domain comprising NORI-011 LCDRs and having at least 90% sequence identity to the VL domain of NORI-011.

[0043] The antibody can comprise a NORI-011 VH domain and a NORI-011 VL domain. Optionally, the antibody is an IgG comprising a NORI-011 heavy chain and a NORI-011 light chain.

[0044] In various embodiments of the application, the % sequence identity shared by the VH and / or VL domains is optionally higher than 90%, for example it can be 95% or higher, 98% or higher or 99% or higher.

[0045] The binding polypeptide can comprise an antibody VH domain produced by recombination of v, d, and j gene segments that are the v, d, and j gene segments that produce the VH domain of any one of NORI-001 to NORI-033. The binding polypeptide can comprise an antibody VL domain produced by recombination of v and j gene segments that are the v and j gene segments that produce the VL domain of any one of NORI-001 to NORI-033. For example, the binding polypeptide can comprise a VH domain produced by recombination of v, d, and j gene segments that produce the VH domain of any one of NORI-001 to NORI-033, and it can comprise an antibody VL domain produced by recombination of v and j gene segments that are the v and j gene segments that produce the VL domain of the antibody.

[0046] Inhibition of MTP-2 enzyme activity can be determined in an in vitro assay for inhibition of cleavage of a MTP-2 substrate by a serine protease to produce a detectable product. Such an in vitro enzyme assay can comprise contacting a polypeptide with MTP-2 or an extracellular domain of MTP-2, and detecting the extent of reduction in production of the detectable product relative to a control assay lacking the polypeptide (which can comprise a negative control polypeptide in its place). The enzyme assay can be performed with a range of concentrations of the polypeptide to produce a dose-response curve from which an IC50 value can be calculated. Thus, inhibitors according to the application can be identified by their dose-dependent inhibition in such an enzyme assay.

[0047] A suitable in vitro enzyme assay is an enzyme assay using MTP-2 and a fluorescent MTP-2 substrate at a final concentration of 50 μΜ. An exemplary substrate is Boc-Gln-Gly-Arg-AMC, which is currently available as Baychem 4016429. MTP-2 in such an assay can have an activity rate of 0.075 U / μl. MTP-2 can be provided as a purified protein in solution, for example an extracellular domain of MTP-2. Thus, an IC50 of a binding polypeptide can be determined in an enzyme assay with a purified extracellular domain of MTP-2 having an activity rate of 0.075 U / μl in the presence of 50 μΜ Boc-Gln-Gly-Arg-AMC fluorescent substrate. A binding polypeptide can have an IC50 of less than 100 nM in such an assay.

[0048] As an alternative to purified ECD, inhibition can be determined in vitro in a cell-based assay (for example using HEK293 cells) with cell surface expressed MTP-2.

[0049] The effect of MTP-2 inhibition (for example, reduction in expression of hepcidin, measurable as a reduction in mRNA of the gene encoding it, hamp) can be further detected in vivo, confirming inhibitory activity and biological relevance.

[0050] In various embodiments, the potency of an inhibitor is quantified according to its IC50 for inhibiting MTP-2 enzyme activity as measured in an in vitro assay, e.g., an assay as described above. Preferably, a binding polypeptide according to the application has an IC50 in an in vitro assay for inhibiting MTP-2 enzyme activity of less than 100 nM, less than 80 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 25 nM, less than 20 nM, less than 15 nM, or less than 10 nM. The IC50 is optionally at least 0.01 nM, at least 0.1 nM, at least 1 nM, at least 2 nM, at least 3 nM, or at least 5 nM.

[0051] Potency can be compared to one or more anti-MTP-2 antibodies described herein for reference. For example, an antibody comprising the VH and VL domains of any one of NORI-001 to NORI-033 can be used as a reference antibody. The reference antibody can be provided as an IgG. For example, the IC50 of a binding polypeptide according to the application can be within 25% or 10% of the IC50 of any one of NORI-001 to NORI-033 (e.g., NORI-003 IgG, NORI-006 IgG, NORI-008 IgG, NORI-009 IgG, or NORI-011 IgG), or its IC50 can be lower than the IC50 of the reference antibody. “Within x%” means that the IC50 of the test binding polypeptide is no more than x% greater and no more than x% less than the IC50 of the reference antibody.

[0052] Inhibitory potency can be compared to the inhibitory potency of aprotinin, a 6500 dalton pan-serine protease inhibitor known to occupy the active site of serine proteases. The IC50 of a binding polypeptide can be similar to or less than the IC50 of aprotinin. The IC50 of a binding polypeptide can be within 50%, within 25%, or within 10% of the IC50 of aprotinin.

[0053] In one embodiment, the assay for MTP-2 inhibition is performed with human MTP-2. In another embodiment, the assay for MTP-2 inhibition is performed with non-human (e.g., mouse, rat, or cynomolgus monkey) MTP-2. Comparing the potency of inhibition of a binding polypeptide in the same enzyme assay using MTP-2 of different species provides an indication of the species cross-reactivity of the binding polypeptide. Generally, species cross-reactivity is desirable because it makes it possible to test a binding polypeptide in vivo in multiple species - for example, preclinical work can be performed in laboratory animals (e.g., mice, rats, or cynomolgus monkeys) prior to clinical work in the target species (e.g., humans). Preferably, a binding polypeptide according to the application is cross-reactive for binding to and inhibiting MTP-2 of multiple species. Preferably, it binds to and inhibits human and mouse MTP-2. More preferably, it binds to and inhibits human, mouse, rat, and cynomolgus monkey MTP-2.

[0054] The IC50of a binding polypeptide in an in vitro assay for inhibition of non-human (e.g., mouse, rat, and / or cynomolgus monkey) MTP-2 enzyme activity can be within 50%, within 25%, or within 20% of the IC50in an in vitro assay for inhibition of human MTP-2 enzyme activity.

[0055] The IC50of a binding polypeptide in an in vitro assay for inhibition of non-human (e.g., mouse, rat, and / or cynomolgus monkey) MTP-2 enzyme activity can differ by less than 100-fold, less than 50-fold, less than 10-fold, less than 5-fold, or less than 2-fold, less than 50-fold, less than 10-fold, less than 5-fold, or less than 2-fold from its IC50in an in vitro assay for inhibition of human MTP-2 enzyme activity.

[0056] Similarly, another measure of species cross-reactivity can be provided by comparing the affinity of a binding polypeptide for MTP-2 of one species to its affinity for MTP-2 of another species. The binding affinities (Kd) can be compared, for example, as determined by surface plasmon resonance. The Kdof a binding polypeptide for non-human (e.g., mouse, rat, and / or cynomolgus monkey) MTP-2 can differ by less than 50-fold, less than 10-fold, less than 5-fold, or less than 2-fold from its Kdfor human MTP-2.

[0057] The ability of a binding polypeptide to compete with a reference molecule for binding to MTP-2 can be determined in vitro. Competition for binding to MTP-2 can be determined in assays using full-length MTP-2, the MTP-2 extracellular domain, the serine protease catalytic domain, and / or other isolated fragments or domains optionally selected based on the region of MTP-2 to which the reference molecule binds. For example, a binding polypeptide according to the application can be one that competes with a serpin polypeptide for binding to MTP-2. A binding polypeptide according to the application can be one that competes with any of the anti-MTP-2 antibodies described herein for binding to MTP-2. For example, an antibody comprising the VH and VL domains of any of NORI-001 through NORI-033 can be used as a reference antibody. The reference antibody can be provided as an IgG. For example, in various embodiments, a binding polypeptide can be one that competes with NORI-003 IgG, NORI-006 IgG, NORI-008 IgG, or NORI-011 IgG for binding to MTP-2. Alternatively, the reference antibody can be provided as an scFv. For example, in various embodiments, a binding polypeptide can be one that competes with NORI-003 scFv, NORI-006 scFv, NORI-008 scFv, or NORI-011 IgG for binding to MTP-2.

[0058] The IC50in a competition assay can be determined. For example, a binding polypeptide can have an IC50of less than 20 nM in a competition assay with a labeled serpin polypeptide for binding to human MTP-2. As shown in Table S, the 58 amino acid mature sequence of a serpin polypeptide can be used in a competition assay.

[0059] Also provided are nucleic acids encoding the binding polypeptides described herein, as well as cells comprising the nucleic acids. In vitro host cells can comprise nucleic acids that are optionally integrated into their cellular (e.g., genomic) DNA or are transiently transfected (e.g., plasmid DNA).

[0060] These and other aspects and embodiments of the application, including methods of producing binding polypeptides, pharmaceutical compositions, and methods of treating patients, are described in greater detail below.

[0061] Clause

[0062] Antibodies against the protease MTP-2 are provided. Inhibition of MTP-2 reduces uptake of dietary iron and reduces release of iron from cellular stores in the body. Inhibitors of MTP-2, such as antibodies against the serine protease domain, can be used to treat iron overload, which is characteristic of diseases such as beta-thalassemia and would otherwise result in toxic accumulation of iron. Combinations of MTP-2 inhibitors with activin receptor ligand traps or with erythropoietin provide additional therapeutic effects.

[0063] The following numbered clauses represent embodiments of the invention and are part of the specification.

[0064] 1. An isolated binding polypeptide that binds to MTP-2 and inhibits the enzymatic activity of the MTP-2, optionally wherein the binding polypeptide binds to the serine protease catalytic domain of MTP-2.

[0065] 2. The binding polypeptide of clause 1, comprising an immunoglobulin domain, wherein the binding site for MTP-2 is formed by loop regions of the immunoglobulin domain.

[0066] 3. The binding polypeptide of clause 2, which is an antibody, optionally a human antibody.

[0067] 4. The binding polypeptide of any preceding clause, wherein the MTP-2 is human MTP-2.

[0068] 5. The binding polypeptide of clause 4, wherein the MTP-2 is human MTP-2 and mouse MTP-2.

[0069] 6. The binding polypeptide of clause 4 or clause 5, which binds human MTP-2 comprising a sequence polymorphism, wherein residue 253 is K or E and residue 736 is V or A.

[0070] 7. The binding polypeptide of any preceding clause, which does not bind to MTP-1 and optionally does not bind to other members of the type II transmembrane serine protease family.

[0071] 8. The binding polypeptide of any preceding clause, which exhibits dose-dependent inhibition of MTP-2 serine protease activity in an enzyme assay using the MTP-2 extracellular domain and a fluorescent MTP-2 substrate at a final concentration of 50 μΜ.

[0072] 9. The binding polypeptide of clause 8, which has an IC50 of less than 100 nM in an enzyme assay against the human MTP-2 extracellular domain and a fluorescent MTP-2 substrate at a final concentration of 50 μΜ.

[0073] 10. The binding polypeptide of clause 8 or clause 9, which has an IC50 of less than 100 nM in an enzyme assay against the mouse MTP-2 extracellular domain and a fluorescent MTP-2 substrate at a final concentration of 50 μΜ.

[0074] 11. The binding polypeptide of clause 10, which has an IC50 in an enzyme assay using the mouse MTP-2 extracellular domain that differs by less than 100-fold from its IC50 in the assay using the human MTP-2 extracellular domain.

[0075] 12. The binding polypeptide of any preceding clause, which exhibits dose-dependent inhibition of MTP-2 serine protease activity in an enzyme assay using HEK293 cell surface expressed human MTP-2 and a fluorescent MTP-2 substrate at a final concentration of 50 mM.

[0076] 13. The binding polypeptide of clause 11, which has an IC50 of less than 100 nM in an enzyme assay using HEK293 cell surface expressed human MTP-2 and a fluorescent MTP-2 substrate at a final concentration of 50 mM.

[0077] 14. The binding polypeptide of any preceding clause, which competes for binding to human and / or mouse MTP-2 with an IgG comprising the VH and VL domains of any one of NORI-001 to NORI-033.

[0078] 15. The binding polypeptide of clause 14, which competes for binding to the serine protease catalytic domain of human and / or mouse MTP-2 with an IgG comprising the VH and VL domains of NORI-003, NORI-006, NORI-008, or NORI-011.

[0079] 16. The binding polypeptide of any preceding clause, which competes for binding to the serine protease catalytic domain of human and / or mouse MTP-2 with a serpin polypeptide.

[0080] 17. The binding polypeptide of clause 16, which has an IC50 of less than 100 nM in a competition assay with a labeled serpin polypeptide for binding to human and / or mouse MTP-2.

[0081] 18. The binding polypeptide of clause 17, which has an IC50 of less than 50 nM in a competition assay with a labeled serpin polypeptide for binding to human and / or mouse MTP-2.

[0082] 19. The binding polypeptide of clause 18, which has an IC50 of less than 20 nM in a competition assay with a labeled serpin polypeptide for binding to human MTP-2.

[0083] 20. The binding polypeptide of any preceding clause, which has an affinity (Kd) for human MTP-2 of less than 50 nM as determined by surface plasmon resonance.

[0084] 21. The binding polypeptide of any preceding clause, which has an affinity (Kd) for mouse MTP-2 of less than 50 nM as determined by surface plasmon resonance.

[0085] 22. The binding polypeptide of clause 21, wherein the Kd for mouse MTP-2 is within 50-fold of the Kd for human MTP-2.

[0086] 23. The binding polypeptide of any preceding Clause, comprising an antibody heavy chain variable (VH) domain obtained by recombination of the germline vdj gene segment set of any one of NORI-001 to NORI-033 shown in Table G and / or an antibody light chain variable (VL) domain obtained by recombination of the germline vj gene segment set of any one of NORI-001 to NORI-033 shown in Table G.

[0087] 24. The binding polypeptide of Clause 23, wherein the VH domain and the VL domain are each obtained by recombination of the germline gene segment set of NORI-001, NORI-002, NORI-003, NORI-004, NORI-005, NORI-006, NORI-007, NORI-008, NORI-009, NORI-010, NORI-011, NORI-012, NORI-013, NORI-014, NORI-015, NORI-016, NORI-017, NORI-018, NORI-019, NORI-020, NORI-021, NORI-022, NORI-023, NORI-024, NORI-025, NORI-026, NORI-027, NORI-028, NORI-029, NORI-030, NORI-031, NORI-032, or NORI-033 shown in Table G.

[0088] 25. The binding polypeptide of any preceding Clause, comprising an antibody heavy chain variable (VH) domain obtained by recombination of the following germline vdj gene segments:

[0089] IGHV3-9*01, IGHD4-17*01, and IGHJ6*02,

[0090] IGHV4-61*01, IGHD3-22*01, and IGHJ5*02,

[0091] IGHV3-49*05, IGHD3-9*01, and IGHJ4*02, or

[0092] IGHV3-13*01, IGHD3-10*01, and IGHJ3*02.

[0093] 26. The binding polypeptide of any preceding Clause, comprising an antibody light chain variable (VL) domain obtained by recombination of the following germline vj gene segments:

[0094] IGLV2-8*01 and IGLJ2*01,

[0095] IGKV1D-33*01 and IGKJ5*01,

[0096] IGKV1D-33*01 and IGKJ4*01, and

[0097] IGKV3D-7*01 and IGKJ1*01.

[0098] 27. The binding polypeptide according to any preceding Clause, comprising a VH domain comprising a set of heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3, and a VL domain comprising a set of light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3, wherein the HCDR set is the HCDR set of any one of NORI-001 to NORI-033 and / or wherein the LCDR set is the LCDR set of any one of NORI-001 to NORI-033.

[0099] 28. The binding polypeptide according to Clause 27, wherein the HCDR set is the CDR set of NORI-003 and the LCDR set is the LCDR set of NORI-003.

[0100] 29. The binding polypeptide according to Clause 27, wherein the HCDR set is the CDR set of NORI-006 and the LCDR set is the LCDR set of NORI-006.

[0101] 30. The binding polypeptide according to Clause 27, wherein the HCDR set is the CDR set of NORI-011 and the LCDR set is the LCDR set of NORI-011.

[0102] 31. The binding polypeptide according to Clause 27, wherein the HCDR set is the CDR set of NORI-008 and the LCDR set is the LCDR set of NORI-008.

[0103] 32. The binding polypeptide according to any preceding Clause, comprising a VH domain having at least 90% amino acid sequence identity to the VH domain of any one of NORI-001 to NORI-033 and / or a VL domain having at least 90% amino acid sequence identity to the VL domain of any one of NORI-001 to NORI-033.

[0104] 33. The binding polypeptide according to Clause 32, comprising

[0105] a VH domain comprising NORI-003 HCDRs and having at least 90% amino acid sequence identity to the VH domain of NORI-003, and

[0106] a VL domain comprising NORI-003 LCDRs and having at least 90% amino acid sequence identity to the VL domain of NORI-003.

[0107] 34. The binding polypeptide of clause 33, comprising a NORI-003 antibody VH domain and a NORI-003 VL domain.

[0108] 35. The binding polypeptide of clause 32, comprising

[0109] a VH domain comprising NORI-006 HCDRs and having at least 90% amino acid sequence identity to the VH domain of NORI-006, and

[0110] a VL domain comprising NORI-006 LCDRs and having at least 90% amino acid sequence identity to the VL domain of NORI-006.

[0111] 36. The binding polypeptide of clause 32, comprising a NORI-011 VH domain and a NORI-011 VL domain.

[0112] 37. The binding polypeptide of clause 36, comprising

[0113] a VH domain comprising NORI-011 HCDRs and having at least 90% amino acid sequence identity to the VH domain of NORI-011, and

[0114] a VL domain comprising NORI-011 LCDRs and having at least 90% amino acid sequence identity to the VL domain of NORI-011.

[0115] 38. The binding polypeptide of clause 32, comprising a NORI-008 VH domain and a NORI-008 VL domain.

[0116] 39. The binding polypeptide of clause 38, comprising

[0117] a VH domain comprising NORI-008 HCDRs and having at least 90% amino acid sequence identity to the VH domain of NORI-008, and

[0118] a VL domain comprising NORI-008 LCDRs and having at least 90% amino acid sequence identity to the VL domain of NORI-008.

[0119] 38. The binding polypeptide of clause 37, comprising a NORI-008 VH domain and a NORI-008 VL domain.

[0120] 39. The binding polypeptide of any one of clauses 1 to 38, wherein the binding polypeptide comprises an antibody constant region.

[0121] 40. The binding polypeptide of clause 39, wherein the binding polypeptide is an IgG antibody.

[0122] 41. The binding polypeptide of clause 40, comprising a human IgG4PE constant region.

[0123] 42. The binding polypeptide of clause 41, comprising a NORI-003 antibody heavy chain and a NORI-003 antibody light chain.

[0124] 43. The binding polypeptide of clause 41, comprising a NORI-006 antibody heavy chain and a NORI-006 antibody light chain.

[0125] 44. The binding polypeptide of clause 41, comprising a NORI-011 antibody heavy chain and a NORI-011 antibody light chain.

[0126] 45. The binding polypeptide of clause 41, comprising a NORI-008 antibody heavy chain and a NORI-008 antibody light chain.

[0127] 46. An isolated antibody comprising a VH domain and a VL domain as defined in any one of clauses 23 to 38.

[0128] 47. An isolated antibody comprising a VH domain and a VL domain, which VH and VL domains are the VH and VL domains of any one of NORI-001 to NORI-033,

[0129] or comprising the VH and VL domains with one or more of the non-germline residues of the framework regions restored to germline.

[0130] 48. The antibody of clause 47, comprising the VH and VL domains of NORI-003 or comprising the VH and VL domains with one or more of the non-germline residues of the framework regions restored to germline.

[0131] 49. The antibody of clause 47, comprising the VH and VL domains of NORI-006 or the VH and VL domains of which one or more non-germline residues of the framework regions are restored to germline.

[0132] 50. The antibody of clause 47, comprising the VH and VL domains of NORI-011 or the VH and VL domains of which one or more non-germline residues of the framework regions are restored to germline.

[0133] 51. The antibody of clause 47, comprising the VH and VL domains of NORI-008 or the VH and VL domains of which one or more non-germline residues of the framework regions are restored to germline.

[0134] 52. An isolated antibody comprising the heavy and light chains of any one of NORI-001 to NORI-033.

[0135] 53. A monoclonal IgG antibody comprising:

[0136] the NORI-003 antibody heavy chain and the NORI-003 antibody light chain,

[0137] the NORI-006 antibody heavy chain and the NORI-006 antibody light chain,

[0138] the NORI-011 antibody heavy chain and the NORI-011 antibody light chain, or

[0139] the NORI-008 antibody heavy chain and the NORI-008 antibody light chain.

[0140] 54. A nucleic acid encoding a binding polypeptide as defined in any one of clauses 1 to 45 or an antibody as defined in any one of clause 46 or clause 53.

[0141] 55. An in vitro host cell comprising a nucleic acid as defined in clause 54.

[0142] 56. A composition comprising a binding polypeptide as defined in any one of clauses 1 to 45 or an antibody as defined in any one of clauses 46 to 53 formulated with a pharmaceutically acceptable excipient.

[0143] 57. The composition of clause 56 for subcutaneous administration.

[0144] 58. A composition comprising a nucleic acid of clause 54 for in vivo gene therapy.

[0145] 59. The composition of any one of clauses 56 to 58 for use in the treatment of the human or animal body by therapy.

[0146] 60. A combination of drugs comprising (i) an MTP-2 inhibitor and (ii) a TGF superfamily ligand antagonist.

[0147] 61. The combination of clause 60 for use in treating iron overload and normalizing erythropoiesis in a patient.

[0148] 62. A method of treating iron overload and normalizing erythropoiesis in a patient comprising administering to the patient (i) an MTP-2 inhibitor and (ii) a TGF family ligand antagonist, wherein (i) and (ii) are administered simultaneously or sequentially.

[0149] 63. The combination of clause 61 or the method of clause 62, wherein the patient has beta- thalassemia (optionally severe beta-thalassemia), MDS, Blackfan Diamond anemia, hemochromatosis type 1, or hemochromatosis type 3.

[0150] 64. The combination or method of any one of clauses 60-63, wherein the antagonist is an Activin II receptor ligand trap.

[0151] 65. The combination or method of clause 64, wherein the antagonist is an Activin II B receptor Fc fusion protein.

[0152] 66. The combination or method of clause 65, wherein the antagonist is roterkirp.

[0153] 67. The combination or method of clause 64, wherein the antagonist is an Activin II A receptor Fc fusion protein.

[0154] 68. The combination or method of clause 67, wherein the antagonist is soltenkirp.

[0155] 69. A combination of drugs comprising (i) an MTP-2 inhibitor and (ii) erythropoietin.

[0156] 70. The combination of clause 69 for use in treating anemia associated with iron overload in a patient.

[0157] 71. A method of treating anemia associated with iron overload in a patient comprising administering to the patient (i) an MTP-2 inhibitor and (ii) erythropoietin, wherein (i) and (ii) are administered simultaneously or sequentially.

[0158] 72. The combination or method of any preceding clause, wherein the MTP-2 inhibitor is a binding polypeptide or antibody as defined in any preceding clause.

[0159] 72. A method of:

[0160] reducing absorption of dietary iron,

[0161] treating iron overload,

[0162] increasing expression of hepcidin in hepatocytes,

[0163] reducing anemia caused by iron overload,

[0164] decreasing serum iron concentration and / or

[0165] decreasing transferrin iron saturation,

[0166] the method comprising administering to the patient a composition according to any one of Clauses 56 to 58.

[0167] 73. The method according to Clause 72, wherein the patient has beta-thalassemia (e.g., severe or moderate beta-thalassemia), 5q-MDS, or RARS.

[0168] 74. The method according to Clause 72 or Clause 73, wherein the method further comprises administering to the patient an antagonist of a TGF family ligand.

[0169] 75. The method according to Clause 74, wherein the antagonist is an Activin II receptor ligand trap.

[0170] 76. The method according to Clause 75, wherein the antagonist is an Activin II B receptor Fc fusion protein.

[0171] 77. The method according to Clause 76, wherein the antagonist is roxadustat.

[0172] 78. The method according to Clause 75, wherein the antagonist is an Activin II A receptor Fc fusion protein.

[0173] 79. The method according to Clause 76, wherein the antagonist is sofosbuvir.

[0174] 80. The method according to Clause 72 or Clause 73, wherein the method further comprises administering to the patient an erythropoiesis stimulating agent, optionally wherein the erythropoiesis stimulating agent is erythropoietin.

[0175] 81. The method according to Clause 72 or Clause 73, wherein the method comprises administering to the patient an additional therapeutic agent, e.g., roxadustat, to reduce iron overload.

[0176] 82. A composition according to any one of Clauses 56 to 58, for use in a method as defined in any one of Clauses 72 to 81.

[0177] 83. Use of a composition according to any one of Clauses 56 to 58 in the manufacture of a medicament for treating a patient, the treatment comprising a method as defined in any one of Clauses 72 to 81.

[0178] 84. A therapeutic agent that reduces iron overload for use in a method of:

[0179] reducing absorption of dietary iron,

[0180] treating iron overload,

[0181] increasing expression of hepcidin in hepatocytes,

[0182] reducing anemia caused by iron overload,

[0183] reducing serum iron concentration and / or

[0184] reducing transferrin iron saturation,

[0185] in a patient, the method comprising administering the therapeutic agent and a composition according to any one of Clauses 56 to 58 to the patient.

[0186] 85. A therapeutic agent comprising erythropoietin (epo) for use in a method of stimulating erythropoiesis in a patient, the method comprising administering the agent and a composition according to any one of Clauses 56 to 58 to the patient.

[0187] 86. A therapeutic agent comprising a TGFp family ligand antagonist for use in a method of promoting red blood cell maturation in a patient, the method comprising administering the agent and a composition according to any one of Clauses 56 to 58 to the patient.

[0188] 87. The method according to any one of Clauses 74 to 81, the composition for use according to Clause 82, the use of a composition according to Clause 83, the therapeutic agent for use according to any one of Clauses 84 to 86, wherein the method comprises administering the therapeutic agent and the composition separately and sequentially to the patient.

[0189] Matriptase-2 (MTP-2)

[0190] MTP-2 is a type II transmembrane trypsin-like serine protease belonging to the type II transmembrane serine protease (TTSP) family. The corresponding gene for MTP-2, TMPRSS6, is located at 22q12.3.

[0191] The typical isoform of MTP-2 (isoform 1) is an 811 amino acid protein with a molecular weight of 90 kDa. It has a conserved structure similar to closely related TTSP family members such as matriptase-1 and enteropeptidase, which consists of a small N-terminal intracellular signal peptide, a signal anchor that functions as a single transmembrane domain, followed by an extracellular structure composed of sea urchin sperm protein, enteropeptidase, and synaptotagmin-like (SEA) domains, a trunk region containing two complement factor Clr / Cl s, sea urchin embryo growth factor, and bone morphogenetic protein (CUB) domains and three low-density lipoprotein receptor (LDLR) class A repeat sequences, and a C-terminal serine protease (SP) domain. The SP domain has a highly conserved catalytic amino acid triad: histidine (617), aspartate (668), and serine (762), which are essential for enzyme function. Figure 2 .

[0192] As with other TTSP members, the correct processing of MTP-2 into a membrane-bound, enzymatically active form is a complex process in which multiple disulfide bonds within the protein and the presence of other endogenous proteins play a key role. MTP-2 is synthesized into the endoplasmic reticulum membrane and transported to the cell surface as an inactive zymogen, where it autocleaves at an arginine residue within a highly conserved activation motif between the CUB2 domain and the serine protease. The MTP-2 serine protease domain contains four essential disulfide bonds, one of which critically links the domain to the membrane-bound trunk.

[0193] The cleaved form of MTP-2 represents the active form and remains predominantly membrane-bound, where it can cleave other membrane-bound targets on the cell surface. However, in vitro overexpression of MTP-2 leads to a “shed” active form of MTP-2 being found in the supernatant of cultured cells. Whether this shed form is part of the natural MTP-2 biology with a function in vivo or is simply a result of overexpression in cell-based systems remains unknown.

[0194] The amino acid sequence of MTP-2 is shown in Table S. For example, human MTP-2 has the amino acid sequence of Uniprot ID Q8IU80, including the N-terminal leader sequence and cytosolic domain, transmembrane domain, and extracellular domain (ECD). The ECD comprises amino acids 84-811 of the full-length protein. A fragment of MTP-2 comprising the ECD (amino acids 78-811) can be recombinantly produced and used in the assays described herein (e.g., in his-tagged form). Production of MTP-2 for use in the assays is detailed in Example 5.

[0195] The binding polypeptides according to the application can bind to and inhibit any one or more or all of MTP-2 expressed on the surface of a cell, isolated MTP-2 ECD and shed soluble MTP-2 ECD.

[0196] Unless the context indicates otherwise, references to MTP-2 herein can be human or non-human (e.g. mouse, rat or cynomolgus monkey). Preferably, the MTP-2 is human MTP-2.

[0197] Due to the complex processing of MTP-2 involving most of the protein structure, mutations in the protein can result in loss of function. Furthermore, there are known TMPRSS6 polymorphisms, such as rs855791, which can result in increased MTP-2 activity and more effective inhibition of hepcidin. The R576A mutation mutates a key arginine necessary for SP domain cleavage and full MTP-2 activation, thus keeping the protein as an inactive zymogen. The S762A mutation mutates a key serine residue in the SP domain catalytic triad and completely abolishes activity. Thus, as self-cleavage requires self-activity, the protein is also kept as an inactive zymogen. The E114K mutant has been described in patients with non-functional MTP-2 expression. This mutation is in the SEA domain, so it is most likely to prevent the protein from being correctly trafficked to the cell surface.

[0198] There are four known variants of MTP-2, covering approximately 92% of the human population:

[0199] Variant 1 (27.4%);

[0200] rs855791 SNP, which results in a single amino acid change of valine to alanine at position 736 (27.2%);

[0201] Variant with V736A and additional K253E variation (25.9%); and

[0202] Variant of K253E alone (11.4%).

[0203] The binding polypeptides will preferably bind to all four such variants, thus being suitable for treating all or most of the human population by inhibiting the MTP-2 variant expressed by the majority of the human population. Thus, the binding polypeptides can bind to human MTP-2 comprising sequence polymorphisms in which residue 253 is K or E and residue 736 is V or A.

[0204] MTP-2 has four known isoforms. Isoform 1, called the canonical isoform, is full length at 811 amino acids and is expressed primarily in the testes. Isoform 2 is the major isoform in the liver and is missing 9 intracellular amino acids at the N-terminus (802 amino acids). This N-terminal region is thought to be involved in the internalization of membrane-bound MTP-2 and thus internalizes more slowly than isoform 1. Isoform 3 is also expressed in the testes, primarily with isoform 1. It has 9 N-terminal amino acids but utilizes an alternative splice variant of exon 10 that drives expression of a truncated form that lacks the SP domain and is thus not functionally active. Isoform 4 shows the same exons as isoform 2 but also has an additional 22 amino acid exon between exons 16 and 17 that disrupts the SP domain function and is also not functionally active. Isoform 4 is thought to be expressed in tissues where isoform 2 is also expressed and because they lack function, isoforms 3 and 4 are thought to be dominant negative to isoforms 1 and 2. Expression of isoform 3 or 4 has been shown to block isoform 2-mediated HJV cleavage.

[0205] The binding polypeptide can bind at least to active isoforms 1 and 2. Optionally, it can bind isoform 3. Optionally, it can bind isoform 4. The binding polypeptide optionally does not bind isoform 3. Optionally, it does not bind isoform 4. Not binding to the inactive isoforms can be beneficial for a therapeutic molecule aimed at inhibiting MTP-2 activity. Binding to the serine protease catalytic domain of isoform 1 and / or isoform 2, and not to isoform 3 and / or isoform 4 is potentially advantageous.

[0206] Binding to MTP-2

[0207] As noted above, MTP-2 is a multi-domain protein and various mutations in the protein are known to result in loss of function. Thus, a binding polypeptide can be generated that recognizes a binding site in the various domains and inhibits the enzymatic activity of the protein.

[0208] The binding polypeptide can bind to the serine protease catalytic domain of MTP-2. It can bind to auto-activated MTP-2. It can bind to the MTP-2 ECD. It can bind to the MTP-2 proenzyme.

[0209] A binding polypeptide that binds to the catalytic domain of a serine protease can be identified as a binding polypeptide that binds to MTP-2 comprising the domain and does not bind to MTP-2 lacking the domain. A "headless" variant of MTP-2 with a C-terminal truncation that deletes the serine protease domain, but still comprises the remainder of the ECD, can be constructed. Binding to the full-length MTP-2 ECD and not to the headless MTP-2 ECD indicates that the binding polypeptide recognizes an epitope in the catalytic domain of a serine protease. Binding can be measured in an HTRF assay or by surface plasmon resonance, example protocols for which are provided herein. Binding to the catalytic domain of a serine protease can also be identified in an enzyme inhibition assay using MTP-2 ECD and headless MTP-2 ECD. A binder that binds to the catalytic domain of a serine protease can exhibit dose-dependent binding to MTP-2 ECD in this assay, but not dose-dependent binding to headless MTP-2 ECD in the assay.

[0210] Binding to cell surface-expressed MTP-2 (e.g., expressed on HEK293 cells) can be detected by fluorescence-activated cell sorting (FACS).

[0211] A binding polypeptide can compete with a serpin polypeptide for binding to MTP-2 (e.g., MTP-2 ECD, e.g., the serine protease catalytic domain thereof).

[0212] Competition between binding polypeptides can also be assayed. For example, a binding polypeptide can compete with an antibody (e.g., IgG or scFv) comprising the VH and VL domains of any of NORI-001 to NORI-033 or an IgG comprising the complete heavy and light chains of any of NORI-001 to NORI-033. It can compete, for example, with NORI-003 scFv. It can compete with NORI-006 scFv. It can compete with NORI-011 scFv. It can compete with NORI-008 scFv.

[0213] Competition between binding polypeptides indicates that they have epitopes in the same region of MTP-2, e.g., both can bind the same domain with overlapping binding footprints.

[0214] IC50 can be calculated in a competition assay as a measure of the ability of a binding polypeptide to inhibit the binding of a reference molecule (e.g., a serpin polypeptide or a NORI antibody) to MTP-2. A binding polypeptide can have an IC50 of less than 100 nM in such an assay. Optionally, the IC50 is less than 50 nM, e.g., less than 20 nM.

[0215] For example, IC50of a competing protease inhibitor polypeptide can be determined in an HTRF competition assay with a directly labeled protease inhibitor polypeptide (e.g., protease inhibitor polypeptide-647) at a concentration of 5 nM, a binding polypeptide at a concentration of 0.3 nM, and human MTP-2 antigen at a concentration of 10 nM or mouse MTP-2 antigen at a concentration of 60 nM. Secondary antibody (e.g., AD0207) can be used at a 1 : 1000 dilution. See Example 8, where the protease inhibitor polypeptide is directly labeled according to the manufacturer’s instructions using the 647 Fast Labeling Kit (Innova Biosciences - 362-0010). To determine IC50of a binding polypeptide competing with a reference NORI antibody, the reference antibody can be directly labeled (e.g., with 647) as above and used in the same protocol, replacing the protease inhibitor polypeptide with the reference antibody.

[0216] Materials and methods for HTRF competition assay

[0217] Antibodies were titrated in HTRF buffer (DPBS (Gibco - 14190144) containing 0.1% BSA (Sigma - A7906) and potassium fluoride 0.53M (Sigma - 60240-250G) at 4x concentration, starting at 200 nM. 5 pL / well of antibody was added to 384 well white plates (Greiner - 784904). Purified proteins of huMTP-2 and moMTP-2 were diluted in HTRF buffer (huMTP-2 = 40 nM and moMTP-2 = 240 nM) at 4x final concentration and plated at 5 pL / well. Anti-MTP-2 mAb in a moIgG1 backbone (NORI-037) was then diluted in HTRF buffer to 1.2 nM 4x final concentration with DELFIA Eu-N1 rabbit anti-mouse IgG antibody (AD0207) at a 1 : 1000 dilution. Finally, 647 labeled protease inhibitor polypeptide (Sigma - A3428) was diluted in HTRF buffer to 20 nM 4x final concentration and plated at 5 pL / well. Plates were incubated for 3 hours or more in the dark at room temperature. Plates were read on an EnVision plate reader using HTRF 100 flash protocol at 1H, 2H, and 3H. (Ex: 340 nm Em1: 620 nm Em2: 665 nm).

[0218] Binding affinity

[0219] The affinity (Kd) of the binding polypeptide for human MTP-2 can be less than 100 nM, less than 50 nM, less than 25 nM, or less than 10 nM. The affinity (Kd) of the binding polypeptide for mouse MTP-2 can be less than 100 nM, less than 50 nM, less than 25 nM, or less than 10 nM. The affinity (Kd) of the binding polypeptide for rat MTP-2 can be less than 100 nM, less than 50 nM, less than 25 nM, or less than 10 nM. The affinity (Kd) of the binding polypeptide for cynomolgus MTP-2 can be less than 100 nM, less than 50 nM, less than 25 nM, or less than 10 nM.

[0220] In some embodiments, the Kd for binding human MTP-2 can be less than 5 nM, e.g., less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, or less than 0.1 nM. The Kd is optionally at least 0.001 nM, e.g., at least 0.005 nM.

[0221] The binding polypeptide can exhibit an affinity (Kd) within the ranges shown in Example 6 (e.g., in Table K or Table W). The Kd of the binding polypeptide for binding MTP-2 can be the same or lower than the Kd of any one of NORI-001 to NORI-033 IgG or scFv (e.g., NORI-009 IgG). Optionally, the Kd is equal to or lower than the Kd of a serpin polypeptide.

[0222] The affinity of the binding polypeptide for MTP-2 can be quantified according to the equilibrium dissociation constant KD of the binding interaction, i.e., the ratio of the association or binding rate (Ka) to the dissociation or disassociation rate (kd). KD, Ka, and kd for antigen binding can be measured using surface plasmon resonance (SPR). Exemplary SPR procedures and conditions are set forth in Example 6.

[0223] Briefly, SPR can be performed at 25 °C by capturing the binding polypeptide on a chip at a concentration of 1 pg / ml at 10 pl / min for 60 seconds (about 35 to 50 RU can be captured), and injecting MTP-2 (analyte) at 30 pl / min for 120 seconds (association time) and monitoring dissociation for 600 seconds. The analyte can be injected at concentrations of 100, 25, 6.25, 1.56, and 0 nM. Sensorgrams for the binding polypeptide are generated, and the data can be fit to a 1 : 1 interaction model (e.g., using Biacore Evaluation Software with global fit for Rmax, ka, kd, and RI = 0).

[0224] Quantitation of affinity can be performed using SPR with monovalent forms of the antigen-binding polypeptide arm, such as an antibody Fab or Fv comprising an antigen-binding site, or a heterodimeric immunoglobulin (e.g., IgG) with a single antigen-binding arm for the antigen in question. Alternatively, it can be convenient to determine the affinity of a bivalent form of the antigen-binding polypeptide arm, such as an IgG comprising a homodimeric antigen-binding arm. SPR can involve coating a dimer of the antigen-binding polypeptide arm onto a biosensor chip (directly or indirectly), exposing the antigen-binding polypeptide arm to a range of concentrations of the antigen in a buffer solution, detecting binding, and calculating the equilibrium dissociation constant KD for the binding interaction. SPR can be performed at 25°C. A suitable buffer solution is 150 mM NaCl, 0.05% detergent (e.g., P20), and 3 mM EDTA, pH 7.6. HBS-P IX (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% polysorbate 20 pH 7.6) with 2.5 mM CaCl2is an example buffer. Binding data can be fitted to a 1 : 1 model using standard algorithms, which can be intrinsic to the instrument used. A variety of SPR instruments are known, such as Biacore™, ProteOn XPR36™ and (Sapidyne Instruments, Inc).

[0225] As described elsewhere herein, isolated purified MTP-2 ECD can be conveniently used in assays, and is a suitable analyte for SPR.

[0226] Cross-reactivity

[0227] Regulatory agencies can require candidate therapeutic molecules to have demonstrated therapeutic efficacy in laboratory animals prior to their entry into human clinical trials. Examples of mouse models of beta-thalassemia and evaluation of binding polypeptides in wild-type mice are demonstrated herein. In order to test a binding polypeptide in such an animal model, it is desirable that the binding agent cross-reacts with the corresponding antigen from one or more non-human mammals. Thus, it can bind non-human MTP-2 as well as human MTP-2.

[0228] One way to quantify the degree of species cross-reactivity of an antigen-binding molecule (or more precisely, its antigen-binding site) is as a fold difference in its affinity for an antigen of one species compared to its affinity for an antigen of another species, such as a fold difference in its affinity for a human antigen compared to a mouse antigen. Affinity can be quantitated as KD, the equilibrium dissociation constant for the binding of an antigen to an antigen-binding molecule. KD can be determined by SPR as described elsewhere herein.

[0229] The fold difference in affinity of the species cross-reactive binding molecule for binding to human and non-human antigens can be 100-fold or less, 50-fold or less, 30-fold or less, 25-fold or less, 20-fold or less, 15-fold or less, 10-fold or less, or 5-fold or less. In other words, the KD of the extracellular domain for binding to a human antigen can be within 30-fold, 25-fold, 20-fold, 15-fold, 10-fold, or 5-fold of the KD of the extracellular domain for binding to a non-human antigen.

[0230] Preferably, the binding affinity for human and non-human antigens is within 10-fold or less, more preferably within 5-fold or 2-fold, of the KD for binding to non-human MTP-2, for example, can be up to 10-fold higher (preferably up to 5-fold or up to 2-fold) or up to 10-fold lower (preferably up to 5-fold or up to 2-fold) than the Kd for binding to human MTP-2, as determined, for example, by surface plasmon resonance.

[0231] A binding molecule can also be considered to be species cross-reactive if the KD for binding to antigens of both species meets a threshold, for example, if the KD for binding to a human antigen and the KD for binding to a non-human antigen are both 10 mM or less, preferably 5 mM or less, more preferably 1 mM or less. The KD can be 100 nM or less, 50 nM or less, 25 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, or 1 nM or less.

[0232] Cross-reactivity can also be identified by the ability of the binding polypeptide to recognize MTP-2 expressed on cells of multiple species, for example, using FACS. HTRF can also be used to determine binding to and cross-reactivity with MTP-2 of multiple species.

[0233] The binding polypeptide can have a measurable ability to block enzymatic activity, as determined by a fluorescent readout using a substrate for MTP-2 from multiple species (e.g., one or more or all of human and mouse, rat, and cynomolgus monkey MTP-2). It can exhibit dose-dependent inhibition of MTP-2 catalytic activity in the assays described herein using human and non-human (e.g., mouse, rat, or cynomolgus monkey) MTP-2.

[0234] While species cross-reactivity for binding to antigens of different species can be advantageous, there remains a need for selectivity of the binding to MTP-2 to avoid unwanted side effects. Thus, in vivo, MTP-2 is preferably the only antigen bound by the antigen binding site of the binding polypeptide. Nonetheless, the binding polypeptide can optionally be engineered to comprise additional binding sites, and an antibody comprising an antibody constant region can, for example, optionally bind to one or more Fc receptors.

[0235] The binding polypeptide optionally does not bind MTP-1 (e.g. human MTP-1). Optionally, it does not bind MTP-3 (e.g. human MTP-3). Optionally, it does not bind other members of the type II transmembrane serine protease family.

[0236] Inhibition of MTP-2

[0237] MTP-2 is primarily expressed on hepatocytes and has a major role in iron metabolism by regulating hepcidin expression in hepatocytes. It is now established that hepcidin (the major regulator of iron homeostasis) expression is controlled by bone morphogenetic protein (BMP) growth factors that bind to type I and type II BMP receptors present on hepatocytes and induce BMP / cascade molecules against the decapentaplegic (SMAD) signaling pathway. Phosphorylation of the Smadl, 5, 8 / Smad4 complex downstream of the BMP receptors increases expression of the HAMP gene encoding hepcidin and increases hepcidin secretion. Hepcidin reduces iron levels in the blood by binding to the iron transporter ferroportin present on duodenal enterocytes, macrophages and hepatocytes, inducing internalization and degradation and thus reducing the amount of iron that enters the blood. MTP-2 is thought to negatively regulate hepcidin expression by selectively cleaving members of the BMPR complex on the surface of hepatocytes and thus silencing the BMP / SMAD signaling. One proposed enzymatic target of MTP-2 is hemochromatosis (HJV), an accessory receptor of the BMPR complex that is required for maximal BMP / SMAD signaling. However, while studies have shown that MTP-2 is able to cleave HJV and produce a specific cleavage product, MTP-2 masked mice show paradoxical reduction in expression of membrane-bound HJV, while TMPRSS6 KO mice show increased levels of cleaved HJV. Recently, studies suggest that MTP-2 most likely cleaves multiple members of the BMPR complex to suppress hepcidin expression.

[0238] According to a tissue-wide scan of TMPRSS6 mRNA expression, there is also low level expression of MTP-2 in the testes, however, the role of MTP-2 here is largely unknown. Due to the localized expression of MTP-2, TMPRSS6 KO mice and humans with loss-of-function MTP-2 mutations show a phenotype where iron levels are greatly elevated and no additional side effects, suggesting that additional roles beyond iron regulation are limited, thus there is little concern for the existence of anti-MTP-2 therapies.

[0239] Accordingly, the present invention proposes inhibiting MTP-2, thereby preventing or reducing cleavage of its downstream substrates, leading to reduced inhibition of hepcidin expression by MTP-2.

[0240] Inhibition of MTP-2 refers to inhibition of the enzymatic activity of MTP-2. MTP-2 is a serine protease, and the inhibitor can be one that inhibits the catalytic serine protease cleavage of its substrate by mature active MTP-2 and / or inhibits the autocatalytic activation of the MTP-2 zymogen by catalytic serine protease cleavage.

[0241] In various embodiments, the binding polypeptide can bind the serine protease catalytic domain of MTP-2. The serine protease catalytic domain contains the enzymatic active site of MTP-2. Inhibition can result from steric hindrance of the enzyme-substrate interaction caused by the binding polypeptide binding to MTP-2 and partially or completely masking the enzymatic active site, thereby reducing substrate binding. Inhibition can alternatively or additionally be caused by the binding polypeptide inducing an inactive conformational change in the serine protease catalytic domain, or biasing the serine protease catalytic domain toward an inactive conformation, whereby its enzymatic activity is reduced. Regardless of the molecular mechanism of inhibition, the ability of the binding polypeptide to inhibit the MTP-2 serine protease activity can be functionally assayed in an enzyme assay.

[0242] Described herein are in vitro assays for inhibiting the serine protease cleavage of a substrate of MTP-2 to produce a detectable product. These include enzyme assays with purified MTP-2 ECD and a fluorescent substrate, and enzyme assays with cell surface expressed MTP-2 and a fluorescent substrate. Each of these assays measures inhibition of the cleavage of a substrate by MTP-2. The fluorescent substrate Boc-Gln-Gly-Arg-AMC can be used, for example, at a final concentration of 50 μΜ. Binding polypeptides that are inhibitors of MTP-2 enzymatic activity can be identified by dose-dependent inhibition of MTP-2 serine protease activity in such enzyme assays.

[0243] In one embodiment, the binding polypeptide has an IC50 of less than 100 nM in an enzyme assay against human MTP-2 ectodomain having an activity rate of 0.075 U / μl in the presence of 50 μΜ Boc-Gln-Gly-Arg-AMC fluorescent substrate. The IC50 can be less than 80 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 25 nM, less than 20 nM, less than 15 nM, or less than 10 nM. The IC50 is optionally at least 0.01 nM, at least 0.1 nM, at least 1 nM, at least 2 nM, at least 3 nM, or at least 5 nM.

[0244] In one embodiment, the binding polypeptide has an IC50 of less than 100 nM in an enzyme assay against non-human (e.g., mouse, rat, or cynomolgus monkey) MTP-2 extracellular domain with 0.075 U / μl activity rate in the presence of 50 μΜ Boc-Gln-Gly-Arg-AMC fluorescent substrate. The IC50 can be less than 80 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 25 nM, less than 20 nM, less than 15 nM, or less than 10 nM. The IC50 is optionally at least 0.01 nM, at least 0.1 nM, at least 1 nM, at least 2 nM, at least 3 nM, or at least 5 nM.

[0245] As noted above, preferably the binding polypeptide is species cross-reactive and thus inhibits MTP-2 from more than one species, e.g., inhibits human and non-human (e.g., mouse, rat, or cynomolgus monkey) MTP-2. Parameters for quantifying cross-reactivity in assays are discussed elsewhere herein.

[0246] In one embodiment, the binding polypeptide exhibits dose-dependent inhibition of MTP-2 serine protease activity in an enzyme assay using HEK293 cell surface expressed human MTP-2 and 50 μΜ final concentration fluorescent MTP-2 substrate. It can have an IC50 of less than 100 nM in the assay. The IC50 can be less than 80 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 25 nM, less than 20 nM, less than 15 nM, or less than 10 nM. The IC50 is optionally at least 0.01 nM, at least 0.1 nM, at least 1 nM, at least 2 nM, at least 3 nM, or at least 5 nM.

[0247] Inhibition of MTP-2 enzyme activity can also be detected in hepatocytes. For example, a binding polypeptide that inhibits MTP-2 can increase hepcidin expression in a liver cancer cell line (with or without bmp stimulation), which can be measured as an increase in hamp mRNA relative to control.

[0248] Similar readouts can be obtained from in vivo assays. Inhibition of MTP-2 enzyme activity in mice administered a binding polypeptide can result in an increase in hamp mRNA, a decrease in serum iron, and a decrease in transferrin saturation (TSAT). A binding polypeptide can be administered to a wild-type mouse (e.g., at 10 mg / kg) to measure these effects. It can increase hamp mRNA in hepatocytes by at least 2-fold within 24 hours of dosing, and this increase can persist for 3 days and preferably 21 days after dosing (e.g., after a single 10 mg / kg dose administered intraperitoneally). A binding polypeptide can decrease serum iron concentration in a mouse. This can also be detected within 24 hours, and preferably persists for 3 days and preferably 21 days after dosing. Example experiments and protocols for measuring hamp mRNA, quantifying serum iron, and TSAT are set out in the Examples.

[0249] Binding polypeptides

[0250] A binding polypeptide according to the present application is a polypeptide molecule with the ability to specifically inhibit and bind MTP-2.

[0251] Many classes of binding polypeptides are known in the art, including classic IgG antibodies and other immunoglobulin domain-based binding proteins (see Binz, Amstutz & Pluckthun, Nature Biotechnology 23(10): 1257 2005. Non-immunoglobulin binding molecules are also known, and binding loops can be engineered into other polypeptide scaffolds such as fibronectin.

[0252] Preferably, a binding polypeptide of the present application comprises an immunoglobulin domain, wherein the binding site for MTP-2 is formed by a loop region of the immunoglobulin domain. A preferred embodiment of a binding polypeptide is an antibody.

[0253] An antibody according to the present application is an immunoglobulin or a molecule comprising immunoglobulin domains, whether naturally occurring or produced partially or wholly by synthesis. An antibody can be an IgG, IgM, IgA, IgD or IgE molecule or an antigen-specific antibody fragment thereof (including but not limited to Fab, F(ab')2, Fv, disulfide linked Fv, scFv, single domain antibody, closed conformation multispecific antibody, disulfide linked scfv, diabody), whether derived from any species that naturally produces antibodies, or produced by recombinant DNA technology; whether isolated from serum, B cells, hybridomas, transfectomas, yeast or bacteria. Antibodies can be humanized using routine techniques. The term antibody encompasses any polypeptide or protein comprising an antibody antigen binding site. An antigen binding site (paratope) is the part of an antibody that binds to and is complementary to an epitope of its target antigen (MTP-2).

[0254] The term "epitope" refers to a region of an antigen that is bound by an antibody. Epitopes can be defined as structural or functional. Functional epitopes are usually a subset of structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes can also be conformational, that is, consist of amino acids that are not linear. In certain embodiments, an epitope can include determinants, that is, chemically active surface groups (such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups) of molecules, and in certain embodiments, can have specific three-dimensional structural characteristics, and / or specific charge characteristics.

[0255] An antigen binding site is a polypeptide or domain that comprises one or more CDRs of an antibody and is capable of binding an antigen. For example, the polypeptide comprises CDR3 (e.g., HCDR3). For example, the polypeptide comprises CDR1 and 2 (e.g., HCDR1 and 2) or CDR1-3 (e.g., HCDR1-3) of a variable domain of an antibody.

[0256] An antibody antigen binding site can be provided by one or more antibody variable domains. In one example, the antibody binding site is provided by a single variable domain, e.g., by a heavy chain variable domain (VH domain) or a light chain variable domain (VL domain). In another example, the binding site comprises a VH / VL pair or two or more such pairs. Thus, an antibody antigen binding site can comprise a VH and a VL.

[0257] An antibody can be a complete immunoglobulin, including constant regions, or can be an antibody fragment. An antibody fragment is a portion of a complete antibody, e.g., comprising the antigen binding region and / or variable region of a complete antibody. Examples of antibody fragments include:

[0258] (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains;

[0259] (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region;

[0260] (iii) an Fd fragment consisting of the VH and CH1 domains;

[0261] (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody,

[0262] (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546; incorporated by reference in its entirety herein), which consists of a VH or VL domain; and

[0263] Other examples of antibodies are H2 antibodies comprising a heavy chain dimer (5'-VH-(optional linker)-CH2-CH3-3') and no light chain.

[0264] Single chain antibodies (e.g., scFv) are useful fragments. Multispecific antibodies can be formed from antibody fragments. Antibodies of the application can suitably take any such form.

[0265] Optionally, a binding polypeptide or antibody immunoglobulin domain thereof can be fused or conjugated to additional polypeptide sequences and / or labels, tags, toxins or other molecules. A binding polypeptide can be fused or conjugated to one or more different antigen binding regions, thereby providing a molecule capable of binding a second antigen in addition to MTP-2. For example, an antibody of the application can be a multispecific antibody, e.g., a bispecific antibody, comprising (i) an antibody antigen binding site for MTP-2 and (ii) another antigen binding site (optionally an antibody antigen binding site, as described herein) that recognizes another antigen.

[0266] Antibodies typically comprise antibody VH and / or VL domains. Isolated VH and VL domains of antibodies are also part of the present application. An antibody variable domain is the portion of an antibody light chain and heavy chain that includes the amino acid sequences of the complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) and framework regions (FRs). Thus, within each VH and VL domain are CDRs and FRs. A VH domain comprises a set of HCDRs, and a VL domain comprises a set of LCDRs. VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain. Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Amino acid positions designated as CDRs and FRs can be defined according to Kabat (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991)) or according to IMGT nomenclature, according to the methods used herein.

[0267] Antibodies may comprise an antibody VH domain, which includes VH CDR1, CDR2, and CDR3 and a frame. Alternatively, they may also comprise an antibody VL domain, which includes VL CDR1, CDR2, and CDR3 and a frame. Examples of antibody VH and VL domains and CDRs according to the invention are listed in Table S. All VH and VL sequences, CDR sequences, CDR groups, HCDR groups, and LCDR groups disclosed herein represent aspects and embodiments of the invention. As stated herein, a “CDR group” includes CDR1, CDR2, and CDR3. Therefore, an HCDR group refers to HCDR1, HCDR2, and HCDR3, while an LCDR group refers to LCDR1, LCDR2, and LCDR3. Unless otherwise stated, a “CDR group” includes HCDR and LCDR.

[0268] As described in more detail in the examples, we isolated and characterized antibodies of particular interest, named NORI-001, NORI-002, NORI-003, NORI-004, NORI-005, NORI-006, NORI-007, NORI-008, NORI-009, NORI-010, NORI-011, NORI-012, NORI-013, NORI-014, NORI-015, and NORI-016. , NORI-017, NORI-018, NORI-019, NORI-020, NORI-021, NORI-022, NORI-023, NORI-024, NORI-025, NORI- 026, NORI-027, NORI-028, NORI-029, NORI-030, NORI-031, NORI-032 and NORI-033 ("NORI-001 to NORI-033").

[0269] In all respects of the invention, unless the context otherwise requires, the antibody may be selected from any of these antibodies, or a subset of NORI-003, NORI-006, NORI-011 or NORI-008.

[0270] This invention covers anti-MTP-2 antibodies having the VH and / or VL domain sequences of all antibodies shown in the appended sequence listing and / or figures, as well as antibodies comprising the HCDR and / or LCDR of those antibodies and optionally having the full heavy chain and / or full light chain amino acid sequences of any anti-MTP-2 antibody disclosed herein.

[0271] CDR sequences can be defined by IMGT or by another method such as Kabat. Unless otherwise stated, references to residues in variable domains or CDRs or framework regions refer to IMGT definitions.

[0272] When an antibody VH domain or VL domain comprises one or more residues in a framework region that are different from the germline gene segment from which it was produced by recombination, the non-germline residue can be retained or can be mutated to a different residue, e.g., it can be restored to the germline residue. The corresponding germline gene segment can be identified as the gene segment that most closely aligns with the sequence of the variable domain, and the corresponding germline gene segment for each of the NORI-001 to NORI-033 VH and VL domains is shown in Table G herein.

[0273] An antibody according to the application can comprise one or more CDRs, e.g., CDR3, and optionally CDR1 and CDR2, as described herein, to form a set of CDRs. The CDR or set of CDRs can be the CDR or set of CDRs of any one of NORI-001 to NORI-033.

[0274] The present application provides an antibody comprising the HCDR1, HCDR2, and / or HCDR3 of any one of the antibodies NORI-001 to NORI-033 and / or the LCDR1, LCDR2, and / or LCDR3 of any one of these antibodies, e.g., a set of CDRs. The antibody can comprise a set of VH CDRs of one of these antibodies. Optionally, it can also comprise a set of VL CDRs of one of these antibodies, and the VL CDRs can be from the same or a different antibody than the VH CDRs.

[0275] The present application also provides a VH domain comprising a disclosed set of HCDRs and / or a VL domain comprising a disclosed set of LCDRs.

[0276] Generally, a VH domain pairs with a VL domain to provide an antibody antigen binding site, but as discussed further below, a VH or VL domain alone can be used to bind an antigen. The NORI-003 VH domain can pair with the NORI-003 VL domain, thereby forming an antibody antigen binding site comprising both the NORI-003 VH and VL domains. Similar embodiments are provided for the other VH and VL domains disclosed herein. In other embodiments, the NORI-003 VH pairs with a VL domain other than the NORI-003 VL. Light chain promiscuity is well established in the art. Likewise, the present application provides similar embodiments for the other VH and VL domains disclosed herein.

[0277] Thus, a VH of any one of the antibodies NORI-001 to NORI-033 can pair with a VL of any one of the antibodies NORI-001 to NORI-033.

[0278] An antibody can comprise one or more CDRs, e.g., a set of CDRs, within an antibody framework. The framework region can have a human germline gene segment sequence. Thus, an antibody can be a human antibody having a VH domain comprising a set of HCDRs in a human germline framework. Typically, the antibody also has a VL domain, e.g., comprising a set of LCDRs in a human germline framework. An antibody "gene segment," e.g., a VH gene segment, a D gene segment, or a JH gene segment, refers to an oligonucleotide having the nucleic acid sequence of that portion of the antibody from which it is derived, e.g., a VH gene segment is an oligonucleotide comprising the nucleic acid sequence corresponding to a polypeptide VH domain from the FR1 to the CDR3 portion. Human V, D, and J gene segments recombine to produce a VH domain, while human V and J segments recombine to produce a VL domain. A D domain or region refers to the diversity domain or region of an antibody chain. A J domain or region refers to the joining domain or region of an antibody chain. Somatic hypermutation can result in an antibody VH or VL domain having a framework region that does not precisely match or align with the corresponding gene segment, but sequence alignment can be used to identify the closest gene segment, and thus the particular combination of gene segments from which a particular VH or VL domain is derived. When aligning antibody sequences with gene segments, antibody amino acid sequences can be aligned with the amino acid sequences encoded by the gene segments, or antibody nucleotide sequences can be aligned directly with the nucleotide sequences of the gene segments.

[0279] An antibody of the application can be a human antibody or a chimeric antibody comprising a human variable region and a non-human (e.g., mouse) constant region. An antibody of the application, e.g., has a human variable region, and optionally also has a human constant region.

[0280] Thus, an antibody optionally includes a constant region or portion thereof, e.g., a human antibody constant region or portion thereof. For example, a VL domain can be attached at its C-terminus to an antibody light chain kappa or lambda constant domain. Similarly, an antibody VH domain can be attached at its C-terminus to all or a portion (e.g., a CH1 domain or an Fc region) of an immunoglobulin heavy chain constant region derived from any antibody isotype (e.g., IgG, IgA, IgE, and IgM) and any of the isotype subclasses (e.g., IgG1 or IgG4). Examples of human heavy chain constant regions are shown in Table S.

[0281] Alternatively, a constant region of an antibody of the application can be a non-human constant region. For example, when an antibody is produced in a transgenic animal (examples of which are described elsewhere herein), a chimeric antibody comprising a human variable region and a non-human (host animal) constant region can be produced. Some transgenic animals produce fully human antibodies. Others have been engineered to produce antibodies comprising a chimeric heavy chain and a fully human light chain. When an antibody comprises one or more non-human constant regions, these can be replaced with human constant regions to provide an antibody that is more suitable for administration to a human as a therapeutic composition, as their immunogenicity is thereby reduced.

[0282] Papain digestion of antibodies produces two identical antigen-binding fragments (also referred to as "Fab" fragments) and an "Fc" fragment (which does not have antigen binding activity but has the ability to crystallize). "Fab" when used herein refers to an antibody fragment, which includes one constant and one variable region of each of the heavy and light chains. The term "Fc region" herein is used to define a C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. An "Fc fragment" refers to the carboxy-terminal portion of heavy chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, which region is also recognized by Fc receptors (FcR) found on certain types of cells. Digestion of antibodies with pepsin produces F(ab')2 fragments, in which the two arms of the antibody molecule remain linked and contain two antigen binding sites. F(ab')2 fragments have the ability to cross-link antigen.

[0283] "Fv" when used herein refers to the minimum fragment of an antibody that retains both antigen recognition and antigen binding sites. This region consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent or covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0284] The antibodies disclosed herein can be modified to increase or decrease serum half-life. In one embodiment, one or more of the following mutations are introduced: T252L, T254S, or T256F to increase the biological half-life of the antibody. The biological half-life can also be increased by altering the heavy chain constant region CH1 domain or CL region to include a salvage receptor binding epitope taken from two loops of the CH2 domain of the Fc region of IgG, as described in U.S. Patent Nos. 5,869,046 and 6,121,022, the modifications described therein incorporated by reference herein. In another embodiment, the Fc hinge region of the antibody or antigen binding fragment of the application is mutated to decrease the biological half-life of the antibody or fragment. One or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment, such that the antibody or fragment has impaired Staphylococcus Protein A (SpA) binding relative to native Fc-hinge domain SpA binding. Other methods of increasing serum half-life are known to those of skill in the art. Thus, in one embodiment, the antibody or fragment is PEGylated. In another embodiment, the antibody or fragment is fused to an albumin binding domain, such as an albumin binding single domain antibody (dAb). In another embodiment, the antibody or fragment is PASylated (i.e., genetic fusion of a polypeptide sequence composed of PAS, which forms uncharged random coil structures with large hydrodynamic volume (XL-Protein GmbH)). In another embodiment, the antibody or fragment is XTENylated / PEGylated (i.e., genetic fusion of a non-precise repeating peptide sequence (Amunix, Versartis) to a therapeutic peptide). In another embodiment, the antibody or fragment is ELPylated (i.e., genetic fusion to ELP repeating sequences (PhaseBio)). These various half-life extending fusions are described in more detail in Strohl, BioDrugs (2015) 29:215-239, e.g., in Tables 2 and 6, incorporated by reference herein.

[0285] Antibody constant regions

[0286] As discussed above, antibodies can be provided in various isotypes and with different constant regions. The Fc region of an antibody is recognized by Fc receptors and determines the ability of the antibody to mediate cellular effector functions, including antibody-dependent cell-mediated cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, and antibody-dependent cellular phagocytosis (ADCP) activity. These cellular effector functions involve the recruitment of cells bearing Fc receptors to the site of target cells, resulting in the killing of the antibody-bound cells.

[0287] In the context of the present application, it is desirable to avoid cellular effector functions, such as ADCC, ADCP and / or CDC. Thus, antibodies according to the present application can lack Fc effector functions, for example they can contain Fc regions that do not mediate ADCC, ADCP and / or CDC, or they can lack Fc regions or be completely devoid of antibody constant regions. The antibodies can have constant regions that are effectorless.

[0288] The antibodies can have heavy chain constant regions that bind one or more types of Fc receptors but do not induce cellular effector functions (i.e. do not mediate ADCC, CDC or ADCP activity). Such constant regions can not be able to bind one or more specific Fc receptors responsible for triggering ADCC, CDC or ADCP activity.

[0289] The antibodies can have heavy chain constant regions that do not bind Fcy receptors, for example the constant region can comprise a Leu235Glu mutation (i.e. where the wild-type leucine residue is mutated to a glutamic acid residue), which can be referred to as an “E” mutation, for example IgG4-E. Another optional mutation to the heavy chain constant region is Ser228Pro (“P” mutation), which increases stability by reducing Fab arm exchange. The heavy chain constant region can be IgG4 comprising both the Leu235Glu mutation and the Ser228Pro mutation. This “IgG4-PE” heavy chain constant region is effectorless. An alternative effectorless human constant region is the disabled IgG1.

[0290] IgG4PE is a preferred antibody isotype of the present application. The binding polypeptide can be an IgG4PE antibody comprising the IgG4PE constant region sequence shown in Table S.

[0291] The antibody constant region can be engineered to have an extended half-life in vivo. Examples include the “YTE” mutation and other half-life extending mutations (Dall’Acqua, Kiener & Wu, JBC 281(33):23514-23524 2006 and WO02 / 060919, incorporated herein by reference). The triple mutation YTE is a substitution of 3 amino acids in the IgG CH2 domain, which mutations provide a tyrosine at residue 252, a threonine at residue 254 and a glutamic acid at residue 256, numbered according to the EU index of Kabat. The YTE modification increases the half-life of the antibody compared to the half-life of a corresponding antibody having a human CH2 wild-type domain, as described in the reference publication. To provide an increased duration of in vivo potency, the antibodies of the present application can include an antibody constant region (e.g. an IgG constant region, e.g. an IgG CH2 domain) having one or more mutations that increase the half-life of the antibody compared to the corresponding wild-type human constant region (e.g. IgG, e.g. IgG CH2 domain). The half-life can be determined by standard methods, as described in WO 02 / 060919.

[0292] Suitable antibody constant regions can be selected according to the genotype of the patient to be treated. For example, a method of increasing erythropoiesis in a human patient can comprise administering a binder (e.g. an antibody) that binds human MTP-2 encoded by a TMPRSS6 nucleotide sequence comprising a SNP selected from:

[0293] rs855791, rs2543519, rs2235324, and rs1421312;

[0294] The binder comprises a constant region selected from:

[0295] (a) a human gamma-4 heavy chain constant region comprising Leu at position 189 as set forth in SEQ ID NO:73 of US20160319017 or Arg at position 289 as set forth in SEQ ID NO:73 of US20160319017; and

[0296] (b) a human gamma-1 heavy chain constant region comprising Asp corresponding to position 204 of SEQ ID NO:42 of US20160319017 or Leu corresponding to position 206 of SEQ ID NO:42 of US20160319017; and wherein

[0297] (i) the human subject comprises a TMPRSS6 nucleotide sequence comprising the selected SNP; and

[0298] (ii) the human patient comprises a constant region gene segment encoding the selected constant region; or the human patient expresses an antibody comprising the selected constant region.

[0299] may be an antibody as described in US20160319017 comprising a constant region as described in the referenced disclosure and / or for use in treating a patient comprising a TMPRSS6 nucleotide sequence polymorphism as described in the referenced disclosure.

[0300] Other example constant regions are shown in Table S.

[0301] Generation and modification of binding polypeptides

[0302] Methods of identifying and making binders to polypeptides, including antibodies, are well known in the art.

[0303] For example, antibodies can be generated using laboratory animals such as mice (including transgenic mice (e.g. Kymouse™, BioMedica, Australia) immunised with MTP-2 or fragments thereof (e.g. recombinant MTP-2 ECD) or their encoding nucleic acids, and the like. HuMab or MeMo ), rat (e.g. ), camelid, shark, rabbit, chicken or other non-human animal, followed by optional humanization of the constant and / or variable regions to produce a human or humanized antibody. In one example, display technologies such as yeast, phage or ribosome display can be used, as will be clear to the skilled person. Standard affinity maturation (e.g. using display technologies) can be performed as a further step after isolation of antibody leads from transgenic animals, phage display libraries or other libraries. Representative examples of suitable technologies are described in US 20120093818 (Amgen, Inc), which is incorporated herein by reference in its entirety, e.g. the methods described in paragraphs

[0309] to

[0346] .

[0304] There are many reasons why it can be desirable to produce variants of the binders, including optimizing the polypeptide sequence for large scale manufacture, facilitating purification, enhancing stability or improving suitability for inclusion in a desired pharmaceutical formulation. Protein engineering work can be performed at one or more target residues in the antibody sequence, e.g. with substitution of one amino acid with an alternative amino acid (optionally, producing a variant containing all naturally occurring amino acids at that position, possibly except Cys and Met), and the effect on function and expression monitored to determine the optimal substitution. In some cases, it is not desirable to substitute a residue with Cys or Met or to introduce these residues into the sequence, as this can create difficulties in manufacture - e.g. by forming new intra- or inter-molecular cysteine-cysteine bonds. When selecting lead candidates and optimizing them for manufacture and clinical development, it is generally desirable to change their antigen binding properties as little as possible, or at least to preserve the affinity and potency of the parent molecule. However, variants can also be produced to modulate key antibody characteristics such as affinity, cross-reactivity or neutralizing potency.

[0305] The antibody can comprise a set of H and / or L CDRs of any of the disclosed antibodies, with one or more amino acid mutations within the disclosed set of H and / or L CDRs. The mutations can be amino acid substitutions, deletions or insertions. Thus, for example, there can be one or more amino acid substitutions within the disclosed set of H and / or L CDRs. For example, there can be up to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 mutations, e.g. substitutions, within the set of H and / or L CDRs. For example, there can be up to 6, 5, 4, 3 or 2 mutations, e.g. substitutions, in HCDR3, and / or up to 6, 5, 4, 3 or 2 mutations, e.g. substitutions, in LCDR3. The antibody can comprise a set of HCDRs, LCDRs or a set of 6 (H and L) CDRs as shown for any of the NORI antibodies herein, or can comprise that set of CDRs with one or two conservative substitutions.

[0306] One or more amino acid mutations can optionally be made in the framework regions of the antibody VH or VL domains disclosed herein. For example, one or more residues that differ from the corresponding human germline segment sequence can be restored to germline. The human germline gene segment sequences corresponding to the VH and VL domains of the exemplary anti-MTP-2 antibodies are indicated in Table G.

[0307] The antibody can comprise a VH domain having at least 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% amino acid sequence identity to the VH domain of any of the antibodies shown in the attached sequence listing, and / or comprise a VL domain having at least 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% amino acid sequence identity to the VL domain of any of those antibodies. Algorithms that can be used to calculate the % identity of two amino acid sequences include, for example, BLAST, FASTA, or the Smith-Waterman algorithm, e.g., with default parameters. Particular variants can include one or more amino acid sequence alterations (additions, deletions, substitutions, and / or insertions of amino acid residues).

[0308] The alterations can be made in one or more framework regions and / or one or more CDRs. The variants are optionally provided by CDR mutagenesis. The alterations typically do not result in loss of function, so that an antibody comprising the altered amino acid sequence can retain the ability to bind MTP-2. It can retain the same quantitative binding ability as the antibody in which the alterations are not made, e.g., as measured in the assays described herein. An antibody comprising the altered amino acid sequence can have improved ability to bind and / or inhibit MTP-2.

[0309] The alterations can include substituting one or more amino acid residues with a non-naturally occurring or non-standard amino acid, modifying one or more amino acid residues into a non-naturally occurring or non-standard form, or inserting one or more non-naturally occurring or non-standard amino acids into the sequence. Examples of the number and location of alterations in the sequences of the application are described elsewhere herein. Naturally occurring amino acids include the 20 “standard” L-amino acids, identified by their standard one-letter codes as G, A, V, L, I, M, P, F, W, S, T, N, Q, Y, C, K, R, H, D, E. Non-standard amino acids include any other residue that can be incorporated into a polypeptide backbone or that results from modifying an existing amino acid residue. The non-standard amino acids can be naturally occurring or non-naturally occurring.

[0310] The term "variant" as used herein refers to a peptide or nucleic acid that differs from a parent polypeptide or nucleic acid by one or more amino acid or nucleic acid deletions, substitutions or additions, but retains one or more specific functions or biological activities of the parent molecule. Amino acid substitutions include alterations wherein the amino acid is replaced with a different naturally occurring amino acid residue. Such substitutions can be classified as "conservative" in which the amino acid residue in the polypeptide is replaced with another naturally occurring amino acid having similar characteristics in polarity, side chain functionality or size. Such conservative substitutions are well known in the art. Substitutions encompassed by the present invention can also be "non-conservative," in which the amino acid residue present in the peptide is replaced with an amino acid having different characteristics, such as substitution of a charged or hydrophobic amino acid with an alanine, or alternatively, in which a naturally occurring amino acid is replaced with a non- conventional amino acid. In some embodiments, the amino acid substitutions are conservative. When used in reference to polynucleotides or polypeptides, the term variant also encompasses polynucleotides or polypeptides that can vary in primary, secondary or tertiary structure compared to a reference polynucleotide or polypeptide, respectively (e.g., compared to a wild-type polynucleotide or polypeptide).

[0311] In some aspects, "synthetic," "recombinant," or "chemically modified" polynucleotide variants or polypeptide variants can be used that are isolated or produced by using methods well known in the art. "Modified variants" can include conservative or non-conservative amino acid changes, as described below. Polynucleotide changes can result in amino acid substitutions, additions, deletions, fusions, and truncations in the polypeptide encoded by the reference sequence. Some aspects use insert variants, deletion variants, or substitution variants with amino acid substitutions, including the insertion and substitution of amino acids and other molecules that are not normally found in the peptide sequence that is the basis of the variant, such as, but not limited to, the insertion of ornithine, which is not normally found in human proteins. When describing polypeptides, the term "conservative substitution" refers to changes in the amino acid composition of a polypeptide that do not substantially alter the polypeptide's activity. For example, a conservative substitution refers to the replacement of a different amino acid residue with an amino acid residue having similar chemical properties (e.g., acidic, basic, positively or negatively charged, polar, or nonpolar, etc.). Conservative amino acid substitutions include replacing leucine with isoleucine or valine, aspartic acid with glutamic acid, or threonine with serine. Conservative substitutions of amino acids that provide functionally similar amino acids are well known in the art. For example, the following six groups each contain amino acids that are conservative substitutions of one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (See also Creighton, Proteins, W. H. Freeman and Company (1984), incorporated by reference in its entirety.) In some embodiments, changes that are single substitutions, deletions, or additions of individual amino acids or small percentages of amino acids can also be considered "conservative substitutions" if the activity of the peptide is not reduced by the change, addition, or deletion. Insertions or deletions are typically in the range of about 1 to 5 amino acids. The selection of conservative amino acids can be selected based on the position of the amino acid to be substituted in the peptide, for example, if the amino acid is on the outside of the peptide and is exposed to solvent, or is on the inside and is not exposed to solvent.

[0312] Amino acids that will substitute the existing amino acids can be selected based on the position of the existing amino acid, including its exposure to solvent (i.e. if the amino acid is exposed to solvent or is present on the outer surface of the peptide or polypeptide as compared to an internally located amino acid that is not exposed to solvent). The selection of such conservative amino acid substitutions is well known in the art, for example as disclosed in Dordo et al., J. Mol Biol, 1999, 217, 721-739 and Taylor et al., J. Theor. Biol. 119 (1986); 205-218 and S. French and B. Robson, J. Mol. Evol. 19 (1983) 171. Thus, conservative amino acid substitutions can be selected that are suitable for amino acids that are external to the protein or peptide (i.e. amino acids that are exposed to solvent), for example but not limited to, the following substitutions can be used: Y is substituted by F, T is substituted by S or K, P is substituted by A, E is substituted by D or Q, N is substituted by D or G, R is substituted by K, G is substituted by N or A, T is substituted by S or K, D is substituted by N or E, I is substituted by L or V, F is substituted by Y, S is substituted by T or A, R is substituted by K, G is substituted by N or A, K is substituted by R, A is substituted by S, K or P.

[0313] In alternative embodiments, encompassed conservative amino acid substitutions that are suitable for amino acids that are internal to the protein or peptide are also selected, for example conservative substitutions that are suitable for amino acids that are internal to the protein or peptide (i.e. amino acids that are not exposed to solvent) can be used, for example but not limited to, the following conservative substitutions can be used: where Y is substituted by F, T is substituted by A or S, I is substituted by L or V, W is substituted by Y, M is substituted by L, N is substituted by D, G is substituted by A, T is substituted by A or S, D is substituted by N, I is substituted by L or V, F is substituted by Y or L, S is substituted by A or T and A is substituted by S, G, T or V. In some embodiments, non-conservative amino acid substitutions are also encompassed within the term of the variant.

[0314] The present application includes methods of producing antibodies comprising VH and / or VL domain variants of the antibody VH and / or VL domains shown in Table S. Such antibodies can be produced by a method comprising:

[0315] (i) providing an antibody VH domain that is a variant of the amino acid sequence of a parent antibody VH domain by adding, deleting, substituting or inserting one or more amino acids in the amino acid sequence of the parent antibody VH domain,

[0316] wherein the parent antibody VH domain is the VH domain of any one of NORI-001 to NORI-033 or a VH domain comprising the heavy chain complementarity determining regions of any one of those antibodies,

[0317] (ii) optionally combining the VH domain thus provided with a VL domain to provide a VH / VL combination, and

[0318] (iii) testing the VH domain or VH / VL domain combination thus provided to identify antibodies having one or more desired characteristics.

[0319] The VH domain can be the VH domain of NORI-003. It can be the VH domain of NORI-006. It can be the VH domain of NORI-011. It can be the VH domain of NORI-008.

[0320] Desired characteristics include binding to human and / or non-human MTP-2. Antibodies having comparable or higher affinity for human and / or mouse MTP-2 relative to the parent antibody can be identified. Other desired characteristics include inhibition in the enzyme assays described herein, and in vivo reduction of serum iron concentration and / or TSAT, and increase in hamp mRNA. Identifying antibodies having desired characteristics can include identifying antibodies having functional attributes described herein, such as their affinity, cross-reactivity, specificity, or neutralization potency, any of which can be determined in assays as described herein.

[0321] When a VL domain is included in the method, the VL domain can be the VL domain of any one of NORI-001 to NORI-033, or can be a variant provided by adding, deleting, substituting, or inserting one or more amino acids in the amino acid sequence of a parent VL domain, where the parent VL domain is the VL domain of any one of NORI-001 to NORI-033 or a VL domain comprising the light chain complementarity determining regions of any one of those antibodies. The VL domain can be the VL domain of the same antibody to which the VH domain belongs. It can be the VL domain of NORI-003. It can be the VL domain of NORI-006. It can be the VL domain of NORI-011. It can be the VL domain of NORI-008.

[0322] The method of producing a variant antibody can optionally include producing copies of the antibody or VH / VL domain combination. The method can further include expressing the resulting antibody. The nucleotide sequences corresponding to the desired antibody VH and / or VL domains can optionally be produced in one or more expression vectors. Suitable methods of expression, including recombinant expression, in host cells are described in detail herein.

[0323] Encoding nucleic acids and methods of production

[0324] Isolated nucleic acids can be provided that encode an antibody according to the application. The nucleic acid can be DNA and / or RNA. Genomic DNA, cDNA, mRNA, or other RNA of synthetic origin, or any combination thereof, can encode the antibody.

[0325] The present application provides constructs in the form of plasmids, vectors, transcriptional or expression cassettes comprising at least one of the above polynucleotides. Exemplary nucleotide sequences are included in the Sequence Listing. Reference to a nucleotide sequence described herein encompasses a DNA molecule having the specified sequence, and encompasses an RNA molecule having the specified sequence in which U is substituted for T, unless context requires otherwise.

[0326] The present application also provides recombinant host cells comprising one or more nucleic acids encoding an antibody. Methods of producing the encoded antibody can include expression from the nucleic acid, for example by culturing a recombinant host cell containing the nucleic acid. The antibody can thus be obtained, and can be isolated and / or purified using any suitable technique, and then used as appropriate. Methods of production can include formulating the product into a composition comprising at least one additional component such as a pharmaceutically acceptable excipient.

[0327] Systems for cloning and expressing polypeptides in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, plant cells, filamentous fungi, yeast, and baculovirus systems, as well as transgenic plants and animals.

[0328] Expression of antibodies and antibody fragments in prokaryotic cells is well established in the art. A common bacterial host is Escherichia coli. Expression in cultured eukaryotic cells is also available to those skilled in the art as a production option. Mammalian cell lines available in the art for expression of heterologous polypeptides include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells, YB2 / 0 rat myeloma cells, human embryonic kidney cells (e.g., HEK293), human embryonic retina cells, and many others.

[0329] The vector can contain appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes, and other sequences as appropriate. The nucleic acid encoding the antibody can be introduced into a host cell. The nucleic acids of the application can be integrated into the genome (e.g., a chromosome) of the host cell. Integration can be facilitated by inclusion of sequences, which promote recombination with the genome. Nucleic acids can be introduced into eukaryotic cells by a variety of methods, including calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses, such as vaccinia virus, or, for insect cells, baculovirus. Introduction of nucleic acids into host cells, particularly eukaryotic cells, can use viral or plasmid-based systems. Plasmid systems can be maintained as episomes, or can be incorporated into the host cell or artificial chromosome. Incorporation can be by random or targeted integration of one or more copies at a single or multiple loci. For bacterial cells, suitable techniques include calcium chloride transformation, electroporation, and transfection using bacteriophage. The nucleic acids can be expressed after introduction, for example, by culturing the host cell under conditions for gene expression, and then, optionally, isolating or purifying the binding polypeptide, such as an antibody.

[0330] Formulation and administration

[0331] Binding polypeptides according to the application, and nucleic acid molecules encoding them, are typically provided in isolated form. The VH and / or VL domains and nucleic acids can be provided in purified form from their natural environment or from their environment of production. Isolated binding polypeptides and isolated nucleic acids will be free or substantially free of material associated with them in nature, such as other polypeptides or nucleic acids with which they are found in vivo, or in the environment in which they are produced, such as a cell culture, when produced by in vitro recombinant DNA techniques. Optionally, an isolated binding polypeptide or nucleic acid (1) is free of at least some other proteins with which it is normally found, (2) is substantially free of other proteins from the same species (e.g., from the same species as the antibody), (3) is expressed by a cell from a different species, (4) has been separated from at least about 50% of the polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is operatively associated with a polypeptide with which it is not naturally associated, either covalently or non-covalently, or (6) does not exist in nature.

[0332] The binding polypeptides or nucleic acids encoding them can be formulated with diluents or adjuvants and remain isolated for practical purposes, e.g., if used to coat microtiter plates for immunoassays, they can be mixed with a carrier, while when used for therapy, they can be mixed with a pharmaceutically acceptable carrier or diluent. Other active ingredients can also be included in therapeutic formulations, as described elsewhere herein. Binding polypeptides can be glycosylated in vivo naturally or by a heterologous eukaryotic cell (such as a CHO cell) system, or can not be glycosylated (e.g., if produced by expression in a prokaryotic cell). Antibodies with modified glycosylation patterns are encompassed by the present application.

[0333] Generally, the isolated product constitutes at least about 5%, at least about 10%, at least about 25%, or at least about 50% of a given sample. The binding polypeptides can be substantially free of proteins or polypeptides or other contaminants found in their natural or production environment that would interfere with their therapeutic, diagnostic, prophylactic, research, or other uses.

[0334] The present application provides therapeutic compositions comprising the binding polypeptides described herein. Therapeutic compositions comprising nucleic acids encoding such binding polypeptides are also provided. Nucleic acids encoding are described in more detail elsewhere herein, and include DNA and RNA, e.g., mRNA. In the therapeutic methods described herein, nucleic acids encoding binding polypeptides and / or cells containing such nucleic acids can be used as a surrogate (or in addition) to compositions comprising the binding polypeptides themselves. Cells containing nucleic acids encoding binding polypeptides, optionally wherein the nucleic acids are stably integrated into the genome, thus represent a drug for patient therapeutic uses. Nucleic acids encoding binding polypeptides can be introduced into human cells derived from the intended patient and modified ex vivo. Administration of cells containing the encoding nucleic acids to a patient provides a reservoir of cells capable of expressing the binding polypeptides, which can provide therapeutic benefits over a longer period of time than administration of isolated nucleic acids or isolated binding polypeptides. Nucleic acids can also be administered directly to a patient for gene therapy. Thus, nucleic acids encoding binding polypeptides can be provided for gene therapy, including introduction of the encoding nucleic acids into cells in a patient such that the nucleic acids are expressed in the patient’s cells and provide a therapeutic effect, examples of which are disclosed herein, including increasing hamp mRNA, decreasing serum iron, decreasing TSAT, and treating diseases and conditions associated with iron overload.

[0335] Compositions can contain suitable agents for modifying the physical form of the dosage unit, for example, coatings, sprays, dusting's, compressions, pellets, films, or capsules. Liquids can optionally be encapsulated using, for example, liposomes or niosomes (vesicles based on non-ionic surfactants). Such preparations for oral administration are suitably thorough mixed with excipients and carried or held in the form by a suitable device or vehicle. The preparations described can contain suitable pharmaceutically acceptable carriers, excipients, and other agents that provide improved transfer, delivery, tolerance, and the like. Many appropriate formulations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, all of which are hereby incorporated by reference. These formulations include, for example, powders, pastes, ointments, creams, waxes, oils, lipids, liposomes (such as LIPOFECTIN™ TM DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures with carbowax. See also, Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311. Compositions can comprise antibodies or nucleic acids in combination with medical injection buffers and / or adjuvants.

[0336] Binding polypeptides or nucleic acids encoding the same can be formulated for the desired route of administration to the patient, for example, in a liquid for injection (optionally an aqueous solution).

[0337] Various delivery systems are known and can be used to administer the pharmaceutical composition of the application. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active agents. Administration can be systemic or local. It is preferred that the antigen-binding molecules are administered by subcutaneous injection. Administration can be patient self-administration, for example, self-injection.

[0338] The pharmaceutical compositions can also be delivered in a vesicle, in particular a liposome (see Langer (1990) Science 249:1527-1533; Treat et al. (1989) Liposomes in the Therapy of Infectious Disease and Cancer, Lopez Berestein and Fidler (eds.), Liss, New York, pp. 353-365; Lopez-Berestein, supra, pp. 317-327; see generally, supra).

[0339] In certain instances, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974). In yet another embodiment, a controlled release system can be placed in proximity to the composition's target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical Applications of Controlled Release, supra, pp. 115-138, 1984).

[0340] Injectable preparations can include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injection, drip infusion, and the like. These injectable preparations can be prepared by known methods. For example, the injectable preparations can be prepared by, for example, dissolving, suspending, or emulsifying the above-described antibody or salt thereof in a sterile aqueous medium or an oily medium conventionally used for injection. As the aqueous medium for injection, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, and the like, which can be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], and the like. As the oily medium, use is made of, for example, sesame oil, soybean oil, and the like, which can be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, and the like. The injection thus prepared can be filled in an appropriate ampoule. The pharmaceutical composition of the present application can be delivered subcutaneously or intravenously with a standard needle and syringe. It is contemplated that the treatment will not be limited to clinical use. Thus, it is also advantageous to use a needleless device for subcutaneous injection. For subcutaneous delivery, a pen delivery device is readily used to deliver the pharmaceutical composition of the present application. Such pen delivery devices can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can be readily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Instead, the disposable pen delivery device is preloaded with the pharmaceutical composition held in a reservoir within the device. Once the pharmaceutical composition within the reservoir is depleted, the entire device is discarded. A number of reusable pen and auto-injection delivery devices have been applied to the subcutaneous delivery of the pharmaceutical composition of the present application. Examples include, but are certainly not limited to, the AUTOPEN® (Owen Mumford, Inc., Woodstock, CT), the BIOJECT® (BIOJECT, Albuquerque, NM), the DEPO-INJ® (Waek Valley International, Inc., Whittier, CA), the EPI-PEN® (Dey, L.P., Jenkintown, PA), the HUMIRA® (Abbott Laboratories, Abbott Park, IL), the HYJEN® (MedicalTM (Owen Mumford, Inc., Woodstock, U.K.), DISETRONIC TM pen (Disetronic Medical Systems, Burgdorf, Switzerland), HUMALOG MIX 75 / 25 TM pen, HUMALOG TM pen, HUMALIN 70 / 30 TM pen (Eli Lilly and Co., Indianapolis, Ind.), NOVOPEN TM I, II, and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR TM (Novo Nordisk, Copenhagen, Denmark), BD TM pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPENT TM , OPTIPEN PRO TM , OPTIPEN STARLET TM , and OPTICLIKT TM (Sanofi-Aventis, Frankfurt, Germany), to name a few. Examples of disposable pen delivery devices for subcutaneous delivery of the pharmaceutical compositions of the application include, but are certainly not limited to, SOLOSTAR TM pen (Sanofi-Aventis), FLEXPEN TM (Novo Nordisk) and KWIKPEN TM (Eli Lilly).

[0341] Advantageously, the pharmaceutical compositions described above for oral or parenteral use are prepared into dosage forms in unit-dose amounts appropriate for the dosage of the active ingredient. Unit-dose forms of such dosage forms include, for example, tablets, pills, capsules, ampoules of injection, suppositories, etc. The amount of the above-described antibody contained is generally about 5 to about 500 mg per unit-dose form of the dosage; especially in the form of an injection, for other dosage forms, it can contain about 5 to about 100 mg and about 10 to about 250 mg of the above-described antibody.

[0342] The binding polypeptide, nucleic acid, or composition comprising the same can be contained in a medical container such as a vial, a syringe, an IV container, or an injection device. In one example, the binding polypeptide, nucleic acid, or composition is extracorporeal and can be in a sterile container. In one example, a kit is provided comprising the binding polypeptide, packaging, and instructions for use in a method of treatment as described herein.

[0343] One aspect of the application is a composition comprising a binding polypeptide or nucleic acid of the application and one or more pharmaceutically acceptable excipients, examples of which are listed above. "Pharmaceutically acceptable" means approved or approvable by a regulatory agency of the Federal or State governments or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans, and more particularly in humans. A pharmaceutically acceptable carrier, excipient, or adjuvant is one that does not destroy the pharmacological activity of the binding polypeptide (e.g., any of the antibody or polypeptide molecules described herein) with which it is combined and that is nontoxic to the patient in whom it is administered, and is neither destructive nor otherwise adverse to the therapeutic activity of the agent when administered at doses sufficient to deliver a therapeutic amount of the agent.

[0344] In some embodiments, the binding polypeptide will be the only active ingredient in the composition according to the application. Thus, the composition can consist of the antibody or it can consist of the binding polypeptide and one or more pharmaceutically acceptable excipients. However, the composition according to the application optionally includes one or more additional active ingredients. Other therapeutic agents that can be desirable for administration with the binding polypeptide or nucleic acid according to the application include other therapeutic agents for iron overload, examples of which are described herein. Any such agent or combination of agents can be administered in combination with the binding polypeptide or nucleic acid according to the application, or provided in a composition, whether as a combined preparation or separate preparations. The binding polypeptide or nucleic acid according to the application can be administered separately and sequentially with another therapeutic agent(s) such as those mentioned, or concurrently and optionally as a combined preparation.

[0345] The various compositions can be administered separately or simultaneously. Separate administration means that two compositions are administered at different times, e.g., at least 10, 20, 30, or 10-60 minutes apart, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 hours apart. The compositions can also be administered at 24 hour intervals, or even longer intervals. Alternatively, two or more compositions can be administered simultaneously, e.g., within less than 10 or less than 5 minutes. In some aspects, compositions administered simultaneously can be administered as a mixture, with or without similar or different time release mechanisms for the components.

[0346] The binding polypeptides and nucleic acids encoding them are useful as therapeutic agents. The patient herein is generally a mammal, often a human. The binding polypeptide or nucleic acid can be administered to the mammal, e.g., by any of the routes of administration described herein. In preferred embodiments, the binding polypeptide is administered by subcutaneous injection.

[0347] Administration is typically carried out in a "therapeutically effective amount", which is an amount sufficient to exhibit the desired effect for which administration is being carried out. The exact amount will depend on the therapeutic objectives and can be ascertainable using known techniques (see, e.g., Lloyd (1999), The Art, Science and Technology of Pharmaceutical Compounding). Treatment regimens, e.g., decisions as to dosages, etc., are within the responsibility of general practitioners and other medical doctors and can depend on the symptoms and / or severity of the disease being treated. A therapeutically effective amount or suitable dosage of a polypeptide or nucleic acid in conjunction can be determined by comparing its in vitro activity and in vivo activity in animal models. Methods for extrapolating effective dosages in mice and other test animals to humans are known.

[0348] In the methods of treatment described herein, one or more doses can be administered. In some cases, a single dose can be effective to achieve long-term benefit. Thus, the methods can comprise administering a single dose of a binding polypeptide, nucleic acid encoding the same, or composition. Alternatively, multiple doses can be administered, typically in sequence and spaced apart by a period of days, weeks, or months. For example, administration can be carried out every 2 weeks, every 3 weeks, or every 4 weeks. Optionally, a binding polypeptide can be administered to a patient once a month or at a lower frequency, e.g., every two months or every three months.

[0349] As used herein, the terms "treat," "treatment," "treating," or "amelioration" refer to therapeutic treatment, wherein the object is to reverse, alleviate, improve, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder. The term "treatment" embraces both reduction or lessening of a condition, disease or disorder, and / or side effects or symptoms associated with the disease or disorder. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of a disease is reduced or halted. That is, "treatment" includes not just the improvement of symptoms or markers, but also a halt or at least slowing of the progression or worsening of symptoms as compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and / or decreased mortality, whether detectable or undetectable. The term "treatment" of a disease also includes providing relief from symptoms or side-effects of the disease (including palliative treatment). Complete cure is not considered for effective treatment. In certain aspects, the methods can also include cure. In the context of the present application, treatment can be prophylactic treatment.

[0350] A long half-life is a desirable characteristic of the binding polypeptides of the present application. A prolonged half-life translates into less frequent administration, which requires fewer injections to maintain a therapeutically effective concentration of the molecule in the bloodstream. The antigen binding molecules of the present application can have an in vivo half-life in humans of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days or more. The antigen binding molecules can have an in vivo half-life in non-human primates, such as cynomolgus monkeys, of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days or more.

[0351] The binding polypeptides can be provided for administration at regular intervals of one week, two weeks, three weeks, four weeks, or one month.

[0352] Therapeutic uses

[0353] The therapeutic indications addressed by the present application include treatment of patients for whom inhibition of MTP-2 activity and / or reduction of iron uptake is beneficial. A particular area of treatment is anemia characterized by high iron loading and deposition and ineffective erythropoiesis. As discussed previously, several forms of anemia are characterized by ineffective erythropoiesis, often accompanied by primary iron overload (driven by inappropriately low hepcidin levels due to high erythropoietic activity) and secondary iron overload primarily due to repeated red blood cell transfusions. Iron overload has a negative impact on organ function and on erythropoiesis, resulting in anemia by increasing high-iron-haem and ROS in erythroid progenitor cells and driving apoptosis, thereby reducing functional red blood cells. Reducing iron can reduce apoptosis and improve a / b globin imbalance, allowing more mature red blood cells to be produced. The reduced availability of iron for the red blood cells results in decreased heme production, increased maturation of red blood cell precursors, and increased hemoglobin levels. Thus, in some cases, the mode of action of the binding polypeptides can be inhibition of the enzymatic activity of MTP-2, resulting in increased hepcidin levels, which leads to iron restriction, normalizing erythropoiesis and improving hemoglobin and red blood cell quality.

[0354] In addition to addressing the toxic effects of iron overload, treatment of anemia can in turn improve cardiac function and reduce fatigue.

[0355] The therapeutic effects according to the present application can include:

[0356] reducing absorption of dietary iron,

[0357] treating iron overload,

[0358] increasing expression of hepcidin in hepatocytes,

[0359] reducing anemia caused by iron overload,

[0360] decreasing serum iron concentration,

[0361] Decreasing transferrin iron saturation,

[0362] Decreasing the need for blood transfusions,

[0363] Decreasing the need for iron chelation therapy,

[0364] Prolonging survival, and / or

[0365] Normalizing erythropoiesis.

[0366] Treatment can be beneficial in many of the disorders discussed herein, including:

[0367] Low risk myelodysplastic syndrome (MDS) with ring sideroblasts (RARS) requiring blood transfusions

[0368] 5q-MDS;

[0369] Transfusion-dependent beta-thalassemia or severe beta-thalassemia;

[0370] Transfusion-independent beta-thalassemia or intermediate beta-thalassemia;

[0371] Hemochromatosis, for example in patients not carrying HJV or hepcidin mutations, or in type 1 or type 3 hemochromatosis;

[0372] Liver cirrhosis;

[0373] Liver steatosis;

[0374] Liver fibrosis, for example in NASH (non-alcoholic steatohepatitis) or ASH patients;

[0375] Blackfan Diamond anemia;

[0376] Pulmonary arterial hypertension;

[0377] Anemia in sickle cell disease ("sickle cell anemia");

[0378] Polycythemia vera;

[0379] Chronic kidney disease-related anemia (CKD).

[0380] Thus, a patient treated according to the application can have one of the above disorders. The method of treatment can comprise administering to the patient a binding polypeptide, nucleic acid or composition as described herein. Examples of formulations and methods of administration are described elsewhere herein. Depending on the disease condition, treatment can be initiated soon after birth, upon diagnosis or upon initiation of transfusion dependency or increase in serum ferritin or at the time iron chelation would otherwise be initiated.

[0381] It is believed that with treatment using anti-MTP-2 binding polypeptides, the increased level of hepcidin will result in a decrease in TSAT levels, with a concomitant decrease in the induction of heme and hematin production in beta-thalassemia or a decrease in the occurrence of ringed sideroblasts in MDS. This will result in less apoptosis of erythroid progenitor cells and more red blood cell counts. While the single cell hemoglobin content will be lower, the total hemoglobin level will be higher due to the increased number of red blood cells. This will decrease the need for red blood cell transfusions.

[0382] Avoiding or reducing the risk of toxic tissue iron overload should increase the overall survival of the patient, i.e. prolong the survival of the treated patient. By avoiding or reducing the transfusion burden, and / or avoiding or reducing the need for iron chelation and / or phlebotomy, the quality of life of the patient should also be improved.

[0383] Treatment can reduce the disease burden and symptoms of the disorder, such as beta-thalassemia, MDS and hemochromatosis associated with iron metabolism, including toxic iron overload, heart failure, liver failure, diabetes and reduced overall survival.

[0384] Interestingly, the genetic polymorphism 736V(A)- >A(G) in Tmprss6 is associated with higher hepcidin levels and has been reported to reduce iron overload and improve liver enzymes in some of these diseases, suggesting that increased hepcidin levels have a positive impact on liver regeneration. Mouse studies have shown a beneficial effect of Tmprss6 and / or hepcidin increase in a mouse model of liver fibrosis.

[0385] Inhibition of MTP-2 can also be used to treat obesity. Folgueras et al. reported that MTP-2 deficiency protects from obesity by regulating iron homeostasis (Folgueras et al., Nat Commun. Apr 10;9(1):1350 2018).

[0386] It is desirable to obtain an effective treatment of the patient without serious side effects. No side effects or only mild side effects are observed.

[0387] Treatment with a binding polypeptide according to the application can be combined with one or more additional therapies, e.g. additional therapeutic agents for the treatment of iron overload. The binding polypeptide can be combined with an activin type II receptor agonist fusion protein, e.g. roxadustat. Since MTP-2 inhibitors present an alternative mode of action to other planned and existing therapies, they potentially provide a synergistic effect on erythropoiesis. Thus, a combination of (i) a MTP-2 inhibitor and (ii) an antagonist of a TGFp superfamily ligand, e.g. a ligand scavenger / trap based on a TGFp superfamily receptor, can result in an advantageous therapeutic effect.

[0388] Various ligand traps act on the TGFβ superfamily to increase late stage erythropoiesis. TGFβ superfamily ligands include activin, GDF-11, and bone morphogenetic proteins (BMPs). Receptor ligand trap molecules can be generated by providing a soluble form of the receptor extracellular domain, which retains the ability to bind one or more of its ligands but does not induce downstream signaling that would otherwise result from normal receptor:ligand interactions. The receptor extracellular domain can be linked to an Fc region to form a fusion protein.

[0389] Preferably, the receptor is an activin II receptor, such as ActIIRA or ActIIRB. The antagonist can be a polypeptide comprising a soluble extracellular domain of an activin II receptor (such as activin receptor IIB (ActRIIB)) fused to an Fc region. Romosozumab is one such molecule, which is currently used to treat anemia in beta-thalassemia and myelodysplastic syndromes. Examples of ActRIIA and ActRIIB ligand traps can also be found in US 7988973 to Acceleron Pharma, which is incorporated by reference herein. Suragani RN et al. previously described “RAP-536”, which is a modified human ActRIIB extracellular domain (native precursor residues 24-131 with L79D substitution) linked to a murine IgG2a Fc domain, which was reported to reduce ineffective erythropoiesis and disease complications in murine beta-thalassemia (Blood 123(25): 3864-3872 2014). Soltenzumab is an example of an activin II type A receptor IgG-Fc fusion protein. Antagonists of other TGFβ family ligands, such as BMPR-Fc fusion proteins, can be similarly generated.

[0390] Alternative antagonists include antibodies against TGFβ superfamily ligands (such as anti-activin antibodies). Anti-activin A antibodies have been described, such as garetosmab.

[0391] ActRIIB-Fc, ActRIIA-Fc, or BMPR-Fc promote maturation of red cell precursors during erythropoiesis, while MTP-2 inhibitors cause iron reduction (normalizing pathologically high iron levels) and thus slow erythropoiesis. From the studies reported herein (see Example 22), we believe that the combination of these two effects results in more efficient production of more mature red blood cells. For example, we demonstrate that the combination of an MTP-2 inhibitor (represented by NORI-11-M) and an activin receptor II ligand trap (represented by ActRIIB-Fc) is more effective than the ligand trap alone in correcting iron overload and associated anemia. The combination with an MTP-2 inhibitor can extend the therapeutic potential of TGFβ superfamily ligand traps by providing greater therapeutic effect in patients with conditions that have already been indicated for TGFβ superfamily ligand traps (e.g., non-transfusion dependent beta-thalassemia patients). The combination with an MTP-2 inhibitor can also extend the therapeutic potential of ligand traps to treat other patient populations such as those with severe beta-thalassemia for whom treatments such as roctadlixcept currently have limited benefit.

[0392] Roctadlixcept was recently approved by the FDA for myelodysplastic syndrome. MDS patients begin to develop iron overload before becoming transfusion dependent, as ineffective erythropoiesis suppresses hepcidin production in the liver, leading to unrestricted intestinal iron uptake. Transfusions in turn exacerbate iron overload. The same is true for transfusion dependent beta-thalassemia patients. In such patients treated with roctadlixcept (or other TGFβ superfamily ligand traps), the therapeutic benefit will be improved by including an MTP-2 inhibitor in the treatment regimen. Combination therapy can be used in any of the therapeutic indications or conditions described herein, for example Blackfan Diamond anemia.

[0393] The MTP-2 inhibitor for use in combination therapy can be a binding polypeptide as described herein, or it can be another type of molecule, such as a nucleic acid inhibitor of TMPRSS6 expression (e.g. an antisense or siRNA molecule targeting TMPRSS6) or a small molecule inhibitor (e.g. 3-amidinophenylalanine-derived proteolysis inhibitors of matriptase-1 and -2 and derivatives). Examples of such inhibitors have been described (Hammami M, Rühmann E, Maurer E, Heine A, Gütschow M, Klebe G, Steinmetzer T (2012) New 3-amidinophenylalanine-derived inhibitors of matriptase. Med Chem Commun 3: 807-813; Pomothy J, Szombath G, Rokonál P, Mátis G, Zs N, Steinmetzer T, Pászti-Gere E (2016) The impact of acute matriptase inhibition in hepatic inflammatory models. Biomed Res Int. https: / / doi.org / 10.1155 / 2016 / 6306984). Thus, a patient treated according to the present application can be a patient who is also treated with an additional therapeutic agent for reducing iron overload. The method can comprise co-administering the binding polypeptide and the additional therapeutic agent, optionally in separate formulations, to the patient. The compositions can be administered sequentially or simultaneously. The same applies to the case where a small molecule or nucleic acid MTP-2 inhibitor is used instead of a binding polypeptide, i.e. the patient can be treated with a combination of MTP-2 inhibitor and additional therapeutic agent, either simultaneously or sequentially. Preferably, the MTP-2 inhibitor and the additional therapeutic agent are provided in separate formulations and administered separately. Typically, sequential administration can be on the same day (optionally separated by a period of a few minutes or a few hours) or on different days.

[0394] Treatment with an MTP-2 inhibitor (such as the binding peptide described herein) can be combined with erythropoietin (epo). Dabepoetin α (commercially known as ARANESP) is a structurally reengineered form of epo with a prolonged drug half-life compared to standard epoα and epoβ proteins. It is used to stimulate erythropoiesis in patients with anemia to increase hemoglobin levels and reduce transfusion requirements. The drug is administered at different doses depending on the severity of anemia; however, in patients with chronic kidney disease-associated anemia (CKD), the recommended starting dose is 0.45 mcg / kg IV / SC every four weeks until hemoglobin levels reach above 10 g / dL, at which point the dose can be reduced. In a small study of patients with intermediate β-thalassemia, ARANESP has shown increased hemoglobin levels (Singer et al. 2011); however, it is generally not a treatment option for patients with β-thalassemia due to an unacceptable increase in RBC cell apoptosis and associated splenomegaly. As the results presented herein show, therapeutic benefits can be obtained by treating patients with the binding peptide according to the invention and with epo. For example, ARANESP and anti-MTP-2 mAb therapy should be co-administered in humans via two separate subcutaneous injections every 2-4 weeks. Based on results obtained in a mouse model of β-thalassemia (see Examples 20 and 21), such a co-treatment strategy should provide a therapeutic improvement in anemia from ARANESP therapy while maintaining spleen size and simultaneously reducing the toxic iron overload provided by anti-MTP2 therapy. Precise combination dosing regimens can be studied and optimized for humans, but will generally involve administering the conjugated peptide according to the invention to the patient, along with erythropoietin (preferably recombinant erythropoietin or a medically approved variant thereof, such as dabetastatin α). Optionally, the conjugated peptide and epo can be administered simultaneously. Alternatively, they can be administered sequentially (on the same day or on different days). Optionally, the conjugated peptide and epo can be administered subcutaneously via separate or combined injections. In some embodiments, the present invention provides treatment of patients with epo and a binding peptide, wherein administration of the binding peptide reduces erythrocyte apoptosis and splenomegaly associated with epo administration in patients suffering from conditions such as β-thalassemia, as mentioned herein. Therefore, the binding peptide can be used to normalize erythropoiesis in patients receiving epo treatment. Attached Figure Description

[0395] Embodiments of the invention will now be described in more detail with reference to the accompanying drawings, in which:

[0396] Figure 1is a diagram of the pathway that regulates the supply of iron for red blood cell production and other cellular functions. Absorption of dietary iron and release of iron recycled from senescent red blood cells ("old RBCs") is regulated by hepcidin, which downregulates ferroportin (FPN), an iron export protein found in the membranes of enterocytes (which take up dietary iron) and macrophages (which take up senescent red blood cells). Inhibition of ferroportin reduces the export of iron from these cells, thereby limiting the supply of new and recycled iron available to the body. Expression of hepcidin from the hamp gene in hepatocytes is regulated by the BMP / SMAD pathway, which is initiated by the binding of the ligand BMP6 to receptors (e.g., HJV and BMPR I-II) on the surface of hepatocytes. Negative feedback from elevated serum iron concentrations can upregulate the BMP / SMAD pathway to increase hepcidin expression and thereby reduce the release of iron through ferroportin. Matriptase-2 (MTP-2) is also present on the surface of hepatocytes and downregulates the BMP / SMAD pathway, presumably by cleaving co-receptors such as HJV, thereby reducing hepcidin expression and enabling more release of iron through ferroportin.

[0397] Figure 2 Domain structure of MTP-2 is shown.

[0398] Figure 3 Full dose response curves of selected anti-MTP-2 antibodies and negative control antibodies in a protein-based enzyme assay using (a) human and (b) mouse MTP-2 are shown. All antibodies are isotype human IgG4PE.

[0399] Figure 4 Inhibition % of antibodies in an enzyme assay using human MTP-2 ECD plotted against inhibition % of antibodies in an enzyme assay using mouse MTP-2 ECD is shown for (a) a first set of antibodies and (b) a second set of antibodies obtained from immunization of transgenic mice.

[0400] Figure 5 Full dose response curves of selected anti-MTP-2 antibodies and negative control antibodies in a cell-based enzyme assay using human MTP-2 ECD are shown. All antibodies are isotype human IgG4PE.

[0401] Figure 6 Results of HTRF assay of antibodies binding to headless human MTP-2 ECD (recombinant MTP-2 ECD lacking the serine protease domain) are shown. IC = isotype control. All antibodies are human IgG4PE.

[0402] Figure 7HTRF assay results showing antibody binding to wild type (a) human, (b) mouse and (c) cynomolgus monkey MTP-2 ECD. IC = isotype control. All antibodies are human IgG4 PE.

[0403] Figure 8 HTRF competition assay results showing selected anti-MTP-2 antibodies or unlabelled aprotinin polypeptide competing for binding to (a) human and (b) mouse MTP-2 ECD. IC = isotype control against labelled aprotinin polypeptide-647. All antibodies are human IgG4 PE.

[0404] Figure 9 (a) hamp mRNA levels and (b) serum iron levels in wild type mice treated with anti-MTP-2 antibodies or controls.

[0405] Figure 10 (a) hamp mRNA levels, (b) serum iron levels, (c) TSAT and (d) MCV in wild type mice treated with 10 mg / kg NORI-010 for 24 hours, 7 days and 14 days or with negative control antibody (single time point).

[0406] Figure 11 (a) hamp mRNA levels, (b) serum iron levels, (c) TSAT and (d) MCV in wild type mice treated with 3 mg / kg NORI-010 for 24 hours, 7 days and 14 days or with negative control antibody (single time point).

[0407] Figure 12 (a) hamp mRNA levels and (b) serum iron levels in mice treated with human or mouse anti-MTP-2 antibodies or their isotype controls.

[0408] Figure 13 Results of time course evaluation of NORI-008 (a) hamp, (b) serum iron, (c) PK following single ip injection at 10, 3 and 1 mg / kg in normal mice.

[0409] Figure 14 Results of evaluation of IgG / kappa anti-MTP2 antibody to reduce serum iron and transferrin saturation at 1 week time point (10 mg / kg IP dose).

[0410] Figure 15 Results of evaluation of three anti-MTP-2 antibodies in fully human IgG4 and mouse IgG1 formats and their ability to reduce serum iron concentration following subcutaneous injection.

[0411] Figure 16(a) serum iron concentration and (b) antibody concentration at day 7 after a single 10 mg / kg sc or ip injection of antibody. In (a), black circles represent the huIgG4PE isotype control, and grey squares represent NORI-010.

[0412] Figure 17 Results of a first in vivo rat study are shown, in which NORI-008 and NORI-010 were dosed at 10 mg / kg IP in groups of 3 wild type Wistar rats.

[0413] Figure 18 shows results of a second in vivo rat study, in which NORI-008, NORI-011, NORI-003 and NORI-006 were each dosed at 10 mg / kg SQ in groups of 5 wild type Han Wistar rats.

[0414] Figure 19 Results of a 2-week study using NORI-010 in a Hbbth3 / + mouse model of intermediate thalassemia are shown. a) Hepcidin mRNA measured from liver samples by qPCR, b) serum iron levels [pg / dL] measured by colorimetric assay and c) transferrin saturation calculated [%].

[0415] Figure 20 shows results of an 8-week study using NORI-011-M in a Hbbth3 / + mouse model of intermediate thalassemia in the presence or absence of erythropoietin. a) Hepcidin mRNA measured from liver samples by qPCR, b) red blood cell count, c) liver tissue iron content per gram of tissue wet weight, d) hemoglobin level, e) hematocrit, f) mean corpuscular volume (MCV), g) red blood cell distribution width (RDW), h) mean corpuscular hemoglobin (MCH), i) number of cells in developmental stages I-V of erythroid lineage cells in the spleen determined by flow cytometry, j) number of cells in developmental stages I-V of erythroid lineage cells in the bone marrow determined by flow cytometry and k) spleen index.

[0416] Figure 21 shows the results of an 8-week study in a Hbbth3 / + mouse model of intermediate thalassemia using NORI-011-M with or without co-treatment with ActRIIB-Fc. NORI-11-M was administered at 10 mg / kg once weekly ip and ActRIIB-Fc was given at 10 mg / kg twice weekly ip. All samples for readout were taken at the end of the 8-week study. a) Hepcidin mRNA measured by qPCR from liver samples, b) Liver tissue iron content per gram of tissue wet weight, c) Red blood cell count (RBC), d) Hemoglobin level (Hb), e) Hematocrit (HCT), f) Mean corpuscular volume (MCV), g) Red blood cell distribution width (RDW), h) Mean corpuscular hemoglobin (MCH), i) Cell numbers in developmental stages I-V of erythroid lineage cells in spleen determined by flow cytometry, j) Cell numbers in developmental stages I-V of erythroid lineage cells in bone marrow determined by flow cytometry and k) Spleen index. Examples

[0417] Here we describe antibodies targeting MTP-2 for the treatment of iron overload diseases. By immunizing transgenic mice that produce antibodies with human variable domains and testing various antibodies in a range of biologically relevant assays, we were able to obtain species-cross-reactive MTP-2 specific monoclonal antibodies (mAbs) that are cross-reactive in vitro and in vivo neutralizers of MTP-2 enzymatic activity. We show that selected mAbs increase hepcidin expression levels in hepatocytes after a single dose. The increase in hepcidin reduces serum iron and transferrin saturation by increasing internalization and degradation of iron transporters. In a Hbb th3 / + In mice, a single dose of 10 mg / kg reduced serum iron and transferrin saturation by 52% and 47%, respectively, at the 2-week time point. Furthermore, by repeated dosing, we reduced the number of reticulocytes in Hbb th3 / + In mice, a single dose of 10 mg / kg reduced serum iron and transferrin saturation by 52% and 47%, respectively, at the 2-week time point. Furthermore, by repeated dosing, we reduced the number of reticulocytes in Hbb

[0418] Example 1 - Generation of an anti-MTP-2 inhibitory antibody panel

[0419] Kymab transgenic mice with antibodies having human variable domains were immunized with MTP-2 using various immunization protocols and antigen formats, and antigen-specific B cells were selected. See Lee et al., Nat Biotechnol 32(4):356-63 2014; WO 2011 / 004192; WO 2011 / 158009; and WO 2013 / 061098. Antibodies were tested for binding to human MTP-2 and mouse MTP-2 and for their ability to inhibit the enzymatic activity of human and mouse MTP-2 in in vitro assays based on protein and based on cells.

[0420] Homogeneous time-resolved FRET (HTRF) and flow cytometry assays were used for initial screening to establish binding of recovered antibodies to purified MTP-2 extracellular domain (ECD), followed by confirmation of binding to cell surface-expressed MTP-2.

[0421] Cross-reactive antibodies were selected for their ability to bind to human and non-human (mouse and cynomolgus monkey) MTP-2 ECD.

[0422] Selected antibodies were then screened in a variety of functional assays to assess their ability to inhibit the enzymatic activity of purified human and mouse MTP-2 ECD in solution and to inhibit the enzymatic activity of human MTP-2 expressed on HEK293 cells, as assessed in enzyme assays containing a chromogenic MTP-2 substrate.

[0423] While many different antibodies were obtained, including examples of antibodies that bind to and inhibit human MTP-2 but not mouse MTP-2 and other antibodies that bind to and inhibit mouse MTP-2 but not human MTP-2, the antibodies listed in Table G below were of particular interest for potential development as cross-reactive inhibitors of MTP-2 activity.

[0424]

[0425]

[0426] Table G. Selected anti-MTP-2 inhibitory antibodies, showing the human germline V, D, and J gene segments from which the VH domains of these antibodies were produced by recombination and the human germline V and J gene segments from which the VL domains of these antibodies were produced by recombination.

[0427] Example 2 - Antibody sequences

[0428] The sequences of the HCDRs, LCDRs, VH domains, and VL domains of each of antibodies NORI-001 through NORI-033 are shown in Table S. The complete IgG4PE heavy chain of each antibody is shown. The complete light chain of each antibody is also shown. Unless the context dictates otherwise, "NORI-001" refers to an antibody having the VH and VL domains shown in Table S for NORI-001. The format of the antibody can be indicated, e.g., "NORI-001 IgG" is an IgG having the NORI-001 VH domain and the NORI-001 VL domain, and "NORI-001 scFv" is an scFv having the NORI-001 VH domain and the NORI-001 VL domain.

[0429] Antibody NORI-002 was determined to have a high risk of a free cysteine propensity in the VH domain. Although believed to be masked by analysis in structural software, this amino acid was mutated to mitigate any potential propensity. Accordingly, a C49C mutation was introduced into NORI-002, and the new antibody comprising this mutation was named NORI-003.

[0430] Antibody NORI-010 was sequence optimized for improved stability and expression by introducing a P124S mutation into the VH domain, resulting in the new antibody NORI-011.

[0431] Example 3 - Inhibition of MTP-2 in a protein-based enzyme assay using MTP-2 ECD

[0432] Antibodies were evaluated for their ability to inhibit serine protease cleavage of a labeled MTP-2 substrate to produce a detectable product in an enzyme assay using human and mouse MTP-2 ECD.

[0433] In this assay, antibodies NORI-001 through NORI-034 all inhibited the enzyme activity of both human and mouse MTP-2. IC50 values for inhibition of human MTP-2 ranged from approximately 1.5 to 55 nM. IC50 values for inhibition of mouse MTP-2 ranged from approximately 0.48 to 40 nM. Table D. Figure 3 .

[0434]

[0435]

[0436] Table D. Potency of antibodies NORI-001 through NORI-034 in inhibiting MTP-2 enzyme activity in protein- and cell-based assays, expressed as IC50 values. When a cell-based assay does not show any value, the antibody was not tested in that assay.

[0437] Cross-reactivity of antibodies was assessed by comparing their inhibition in the assay using human MTP-2 ECD versus their inhibition in the assay using mouse MTP-2. By plotting the percent inhibition values from both assays against each other, it was observed that some antibodies were specific for human MTP-2 while others were specific for mouse MTP-2, and there was varying degrees of inhibition for both human and mouse MTP-2. Figure 4 .

[0438] Materials and methods for protein-based enzyme assays

[0439] For the initial assay, positive and negative controls, aprotinin (Sigma-A3428) and a non-MTP-2 binding huIgG4PE isotype antibody, respectively, were serially diluted 1 :3 in assay buffer (200 mM Tris HCI and 1 mg / mL BSA, pH 9.0) to a 2x final concentration with a starting concentration of 200 nM. In later assays, the positive and negative controls used were 10 nM of NORI-008 huIgG4PE and a non-MTP-2 binding huIgG4PE isotype antibody, respectively.

[0440] Twenty μΐ of titration controls and diluted antibodies were plated in 384 well solid white plates (Alpha plates - 6005350) and 10 μΐ of human or mouse MTP-2 ECD protein was plated on top with a final concentration of 0.5 or 2 mg / ml, respectively, due to differences in enzyme activity. The plates were then covered and incubated at room temperature for 30 minutes. Ten μΐ of fluorescent MTP-2 peptide substrate, Boc-Gln-Gly-Arg-AMC (Bachem AG-4016429.0050), was then added to each well at a final concentration of 50 μΜ in assay buffer. The enzyme reaction was allowed to proceed at room temperature and then read for fluorescent activity on a plate reader (Envision) at 30 minutes, 1 hour, and 2 hours with an excitation wavelength of 360 nm and an emission wavelength of 460 nm. Upon cleavage of the substrate peptide by the enzyme activity of MTP-2, the 7-amido-4-methylcoumarin (AMC, MCA, or NHMec) moiety is liberated from the C-terminus. AMC is a fluorophore incorporated at the C-terminus of carboxypeptidase substrates. The released coumarin can then be excited using a spectrophotofluorimeter by exciting at 360-380 nm and detecting emission at 440-460 nm. The positive and negative controls were taken as the average of 16 wells of aprotinin / NORI-008 IgG and huIgG4PE isotype at a final concentration of 200 nM, respectively. Log curves were generated by inputting the inhibition values into graphpad software and IC50 values were generated using the nonlinear regression parameters and log(inhibitor) versus response-variable slope (four parameters) equation.

[0441] Details of the generation of the antigen reagents are described in Example 5.

[0442] Example 4 - Inhibition of MTP-2 in a cell-based enzyme assay using cell surface expressed MTP-2

[0443] Antibodies were evaluated for their ability to inhibit serine protease cleavage of a labeled MTP-2 substrate to produce a detectable product in an enzyme assay using cell surface expressed human MTP-2.

[0444] In this assay, antibodies NORI-008 through NORI-010, NORI-012 through NORI-014, and NORI-017 through NORI-034 all inhibited the enzymatic activity of human MTP-2. The range of IC50 values for inhibition of human MTP-2 was from about 0.083 nM to 17 nM. Table D. Figure 5 .

[0445] In this cell-based assay, MTP-2 is expressed by the cell and displayed on the cell surface, where it undergoes autoactivation to produce activated MTP-2 that cleaves its substrate. Inhibitors in this assay can act through a variety of molecular mechanisms. For example, an inhibitor that shows inhibition in this assay can bind to and inhibit the proenzyme form of MTP-2 from converting to the autoactivated form of MTP-2 Figure 2 ), thus preventing the formation of the activated form of MTP-2 that would otherwise cleave its substrate in this assay, and / or an inhibitor in this assay can bind to and inhibit the activated form of MTP-2.

[0446] The ability of antibodies NORI-001 through NORI-034 to inhibit cell surface expressed MTP-2 demonstrates their activity against MTP-2 in a cell-based environment where the target antigen undergoes expression and activation on a cell, which is characteristic of in vivo conditions.

[0447] Materials and methods for cell-based enzyme assays

[0448] All antibodies for screening as well as positive and negative controls were serially diluted 1 :3 in Expi293 medium (A1435101) - serum free medium (pH 8) with high glucose and GlutaMAX - 2x final concentration, starting at 200 nM. 12.5 μΙ antibody (purified protein A) and controls were plated into 96 well solid white plates (Corning - CLS3917-100EA) and 25 μΙ cells (6250 cells / well) in Expi293 medium were top dispensed. Then 12.5 μΙ fluorescent MTP-2 peptide substrate (Bachem AG - 4016429.0050) was added to each well at a final concentration of 50 μΜ in Expi293 medium. The enzyme reaction was allowed to proceed overnight in an incubator at 37°C and 5% C02. The next day, the fluorescence activity was read on a plate reader (Envision) at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. The positive and negative controls were taken as the average of 4 wells of a final concentration of 200 nM of aprotinin polypeptide and huIgG4PE isotype, respectively.

[0449] For the initial assay, the positive control was aprotinin polypeptide (Sigma-A3428). For the subsequent assays, the positive control was NORI-008 huIgG4PE. A non-MTP-2 binding huIgG4PE isotype antibody was used as a negative control.

[0450] Details of the generation of the antigen reagent are described in Example 5.

[0451] Example 5 - Preparation of antigen material

[0452] Generation of constructs for protein expression

[0453] To generate purified proteins for the assays described herein, we used DNA sequences encoding:

[0454] • extracellular domain (ECD) aa 77-855 of WT human matriptase-1 (uniprot seq ID no: Q9Y5Y6)

[0455] • wild type (WT) human MTP-2 (uniprot seq ID no: Q8IU80) aa 78-811

[0456] • WT mouse MTP-2 (uniprot seq ID no: Q9DBI0) aa 80-811

[0457] • WT rat MTP-2 (NCBI seq ID: XP006242057.1) aa 80-811

[0458] • Cynomolgus MTP-2 (uniprot seq ID no: A0A2K5VAP0) aa 73-800

[0459] The above amino acid sequences are also provided in Table S as reference.

[0460] These coding sequences were fused to a C-terminal His-tag and N-terminal leader sequence, codon-optimized for mammalian expression and expressed.

[0461] For the production of MTP-2 antigens, expression can be greatly improved by co-expression with untagged moHAI-2 ECD antigen followed by purification of the His-tagged antigen over a nickel column. For this purpose, DNA encoding WT mouse HAI-2 (uniprot seq ID no: Q9WU03) aa 28-197 was fused to a N-terminal immunoglobulin leader sequence, codon-optimized for mammalian expression and expressed.

[0462] DNA sequences were cloned into the pTT5 protein expression vector under the control of the CMV promoter using the restriction sites Nhel and Aatll. Expression plasmids were transfected into CHO-3E7 cells using PEI transfection reagent.

[0463] Generation of full-length antigen constructs for stable cell line generation

[0464] For the purpose of screening for MTP-2 specific antibodies, stable cell lines expressing the relevant antigens were generated.

[0465] Full length DNA sequences encoding WT human MTP-2 (uniprot seq ID no: Q8IU80) amino acids (aa) 1-811, WT mouse MTP-2 (uniprot seq ID no: Q9DBI0) aa 1-811, each fused to N-terminal eGFP and C-terminal flag tag (DYKDDDDK) were codon optimized for mammalian expression. The process was repeated for untagged cynomolgus monkey MTP-2 (uniprot seq ID no: A0A2K5VAP0) aa 1-800. The DNA sequences were cloned into expression vectors under the control of a CMV promoter and flanked by 3’ and 5’ piggyBac specific terminal repeats which facilitate stable integration into the cell genome (see: “A hyperactive piggyBac transposase for mammalian applications”; Yusa K., et al., Proc. Natl. Acad. Sci. U S A., 108(4):1531-6, January 25, 2011). The CMV promoter expression vectors contained a puromycin selection cassette to facilitate stable cell line generation.

[0466] Subtype 2: WT human MTP-2 aa 10-811 and WT mouse MTP-2 amino acids 13-811 in untagged form were generated by PCR directed mutagenesis from the constructs described above and re-cloned into the same expression vectors as previously described. Subtype 2: K253E, V736A and K253E+V736A human WT MTP-2 (uniprot seq ID no: Q8IU80) sequence aa 10-811 variants in untagged form were also generated by PCR directed mutagenesis and re-cloned into the same expression vectors as previously described.

[0467] Full length DNA sequences encoding WT human Matriptase-1 (MTP-1) (uniprot seq ID no: Q9Y5Y6) aa 1-855, WT human Matriptase-3 (MTP-3) (uniprot seq ID no: Q7RTY8) aa 1-854 with C-terminal His tag fusions and untagged WT mouse HAI-2 (uniprot seq ID no: Q9WU03) aa 1-252 were codon optimized for mammalian expression and cloned into the same expression vectors as previously described using a CMV promoter. The expression vectors for MTP-1 and MTP-3 contained a puromycin selection cassette while the expression vector for moHAI-2 contained a neomycin selection cassette to facilitate dual stable cell line generation.

[0468] Generation of stably transfected Hepa 1-6, CHO and HEK-293 cells expressing MTP-1, MTP-2 and MTP-3 antigens

[0469] To generate a human embryonic kidney (HEK)-293 cell line expressing WT human MTP-2 eGFP / Flag tagged aa 1-811, the CMV promoter expression plasmid was co-transfected with a plasmid encoding piggyBac transposase into human embryonic kidney (HEK)-293 cells using FreeStyle Max transfection reagent (Invitrogen) according to the manufacturer's instructions.

[0470] To generate a Chinese hamster ovary (CHO) cell line expressing WT human MTP-2 eGFP / Flag tagged aa 1-811, the CMV promoter expression plasmid was co-transfected with a plasmid encoding piggyBac transposase into Chinese hamster ovary (CHO) cells using FreeStyle Max transfection reagent (Invitrogen) according to the manufacturer's instructions.

[0471] To generate a cell line expressing untagged WT human MTP-2 aa 10-811 and WT mouse aa 13-811 MTP-2, the CMV promoter expression plasmid was co-transfected with a plasmid encoding piggyBac transposase into HEK293. The WT human aa 10-811 and WT mouse aa 13-811 MTP-2 untagged construct was also co-transfected with a plasmid encoding piggyBac transposase into the Hepa 1-6 cell line using FreeStyle Max transfection reagent (Invitrogen) according to the manufacturer's instructions.

[0472] To generate a cell line expressing His tagged WT human MTP-1 aa 10-855 and WT human MTP-3 aa 1-854, the MTP-1 and MTP-3 CMV promoter expression plasmids were co-transfected with a WT moHAI-2 expression plasmid and a plasmid encoding piggyBac transposase into the HEK293 cell line using FreeStyle Max transfection reagent (Invitrogen) according to the manufacturer's instructions.

[0473] Twenty-four hours post-transfection, the media was supplemented with puromycin (2.5 pg / mL) or G418 (1 mg / mL) or both puromycin (2.5 pg / mL) and G418 (1 mg / mL) and grown for at least two weeks to select stable cell lines. Cell culture media was changed every 3-4 days. Following selection, the HEK293 cell line expressing WT human MTP-2 eGFP / Flag-tagged aa 1-811 was serially diluted into single cell clones for maximum expression. This was repeated for the CHO cell line expressing WT and S762A human MTP-2 eGFP / Flag-tagged aa 1-811. Expression of flag-tagged human or mouse MTP-2 constructs on cells was assessed by flow cytometry using anti-flag APC-conjugated antibody (Biolegend-637308), while expression of his-tagged human MTP-1, human MTP-3, and human and mouse MTP-2 constructs was assessed by flow cytometry using untagged anti-His primary antibody (Abeam-ab18184) followed by goat anti-mouse 647-conjugated secondary antibody (Citeab-115-605-071). Following selection, stable HEK293 cells expressing untagged WT human MTP-2 and WT mouse MTP-2 and stable Hepa 1-6 cells expressing untagged WT human MTP-2 and WT mouse MTP-2 were FACS sorted for high expression. Expression of untagged human, mouse, and cynomolgus MTP-2 constructs (including human and mouse mutant and human variant constructs) on cells was assessed by flow cytometry using APC-conjugated anti-MTP-2 antibody.

[0474] Complete HEK293 and Hepa 1-6 media consisted of Dulbecco’s Modified Eagle Medium (Gibco) supplemented with 10% v / v fetal bovine serum (Gibco). Adherent CHO cells were cultured in Ham’s F-12 Nutrient Mix (Gibco) supplemented with 10% v / v fetal bovine serum (Gibco) during transfection. Following transfection, antigen-expressing CHO cells were grown in suspension in complete CHO-S media consisting of CD-CHO media supplemented with 8 mM Glutamax (Gibco). CHO-3E7 cells used here for expression included the pTT5 vector system and CHO EBNA 1 cells available from the National Research Council of Canada, but other CHO cell lines can also be used.

[0475] Example 6 - Surface plasmon resonance assay of binding affinity and kinetics

[0476] Dissociation rate screening was performed by surface plasmon resonance (SPR) using a Biacore 8K system (GE Healthcare). Anti-human Fc mix (about 1000 RU) was immobilized in active and reference channels in HBS-P+ buffer (GE BR100671) pH 7.4. Anti-MTP-2 huIgG4 PE antibodies were then captured on the active channel only at 10 μl / min for 60 seconds at a concentration of 1 μg / ml (about 35 and 50 RU captured). MTP-2 ECD protein analytes were then injected at 30 μl / min for 120 seconds (association time) at concentrations of 100, 25, 6.25, 1.56, and 0 nM and dissociation was monitored for 600 seconds. A multi-cycle kinetic model was used with all 8 channels. The reference and background were subtracted from the sensorgrams of each antibody and the data were fitted using a 1:1 interaction model in the Biacore Evaluation software. Rmax, ka, kd were globally fitted, RI = 0. The range of affinities (Kd) for the tested antibodies was about 0.012 nM to 8.5 nM. Table K.

[0477]

[0478]

[0479] Table K. Antibody: antigen binding kinetics of anti-MTP-2 antibodies determined by surface plasmon resonance. * indicates that the kd (dissociation rate) of the antibody was too slow and could not be determined with the assay setup. True KD can be < the value indicated.

[0480] Binding was also evaluated using the above SPR method with a "headless" huMTP-2 ECD 78-576aa instead of the full ECD. The headless ECD corresponds to the MTP-2 ECD without the serine protease domain. The amino acid sequence of the "headless" huMTP-2 ECD 78-576aa protein corresponds to the human matriptase-2 masked ECD protein his tag set forth in SEQ ID S. No binding of any of the 7 antibodies against the headless protein was detected, indicating that the epitope of all these antibodies is in the serine protease domain.

[0481]

[0482] Table W. Antibody: antigen binding kinetics of cross-reactive anti-MTP-2 antibodies

[0483] NORI-011, NORI-008, NORI-003 and NORI-006 were found to have cross-reactive binding to human, mouse, cynomolgus and rat MTP-2 ECD proteins. The sequences of these proteins used in SPR are given by the human matriptase-2 ECD protein his tag, mouse matriptase-2 ECD protein his tag, cynomolgus matriptase-2 ECD protein his tag, rat matriptase-2 ECD protein his tag and human matriptase-2 masked ECD protein his tag in the sequence listing S. Since none of the four antibodies detected binding to the human matriptase-2 masked ECD protein his tag, this indicates that the binding is not to the his tag, and that the epitope of all antibodies is in the serine protease domain. The true KD value for NORI-008 to human or mouse MTP-2 could not be accurately measured due to very slow off rates.

[0484] Example 7 - Binding to MTP-2 serine protease domain

[0485] SPR analysis of the antibody panel in Example 6 showed no detectable binding to a truncated "headless" huMTP-2 protein that lacks the serine protease domain. This indicates that these antibodies bind to the serine protease domain of MTP-2. Such antibodies can be expected to block MTP-2 mechanically by blocking or distorting the serine protease domain active site, thus preventing cleavage of substrates.

[0486] NORI-003, NORI-006, NORI-008 and NORI-011 were assayed for HTRF binding to this headless protein and no detectable binding was confirmed Figure 6 ), whereas binding was detected for all human, mouse and cynomolgus MTP-2 proteins without C-terminal truncation Figure 7 . This again indicates that the binding epitope of these inhibitory antibodies is in the serine protease domain. Another antibody, NORI-036, binds to both the headless and intact ECD, indicating that it recognizes a binding site on MTP-2 outside of the serine protease domain.

[0487] Example 8 - Competition with serpin polypeptides

[0488] The protease inhibitor is a broad-spectrum serine protease inhibitor and is known to occupy the active site of serine proteases. The HTRF competition assay against the labeled protease inhibitor demonstrated that NORI-003, NORI-006, NORI-008 and NORI-011 bind at the same site or in its vicinity and compete with the protease inhibitor for binding to the human MTP-2 protein. For NORI-003 and NORI-006, this competition was weak despite potent enzyme inhibition IC50values against huMTP-2, suggesting that the epitope against which these clones are directed is likely close to the active site but not identical to the binding site of the protease inhibitor. This was confirmed by their lack of competition with the protease inhibitor for moMTP-2, while the IC50values in the enzyme assay were similar to NORI-011. NORI-008 showed very low Kdvalues and IC50values in the SPR and protease inhibitor competition assays, respectively, indicating that its binding site is identical or closely overlapping with the binding site of the protease inhibitor. Figure 8 Table L.

[0489]

[0490] Table L. Antibody IC50values for enzyme inhibition and competition with protease inhibitor for human and mouse MTP-2.

[0491] Materials and methods for HTRF competition assay

[0492] Antibodies were titrated in HTRF buffer (DPBS (Gibco-14190144) containing 0.1% BSA (Sigma-A7906) and potassium fluoride 0.53M (Sigma-60240-250G)) at 4x concentration, starting at 200nM. 5μL / well of antibody was added to 384 well white plates (Greiner-784904). Purified proteins of huMTP-2 and moMTP-2 were diluted into HTRF buffer (huMTP-2 = 40nM and moMTP-2 = 240nM) at 4x final concentration and plated at 5μL / well. Anti-MTP-2 mAb in a moIgG1 backbone (NORI-037) was then diluted to 1.2nM 4x final concentration in HTRF buffer with DELFIA Eu-N1 rabbit anti-mouse IgG antibody (AD0207) at 1:1000 dilution 4x final concentration. Finally, 647 labeled protease inhibitor (Sigma-A3428) was diluted to 20nM 4x final concentration in HTRF buffer and plated at 5μL / well. Plates were incubated for 3 hours or more in the dark at room temperature. Plates were read on an EnVision plate reader using HTRF 100 flash scheme at 1H, 2H and 3H. (Ex: 340nm Em1: 620nm Em2: 665nm).

[0493] Example 9 - Progression to in vivo studies

[0494] MTP-2 has been validated as a target for addressing iron overload anemia by increasing hepcidin concentration through various models and concepts. Human genetics has shown that a deficiency or blockade of MTP-2 activity can increase hepcidin levels and thus reduce iron overload in preclinical models and clinical interventions. For example, Tmprss6 knockout mice are viable but are characterized by high hepcidin levels, so iron restriction leads to body hair loss but not around the head area, producing a temporary so-called "masking" phenotype. Crossing these mice with beta thalassemia mice with hemizygous beta globin chain deletion, the red blood cell count and hemoglobin can be improved due to the increase in hepcidin seen in these crosses (Nai et al., 2012). Furthermore, in humans, mutations of the Tmprss6 gene cause a rare form of anemia in which hepcidin levels are abnormally high, such that patients suffer from iron deficiency that cannot be corrected by giving more iron (Iron Refractory Iron Deficiency Anemia - IRIDA) (Lenoir et al., Blood 117:647-650 2011; Nai et al., 2012 supra).

[0495] Inhibition of MTP-2 activity suggests that antibodies can be able to increase hepcidin expression and thus prevent iron overload, which is the main cause of morbidity and mortality in B-thalassemia. Antibodies that demonstrate this activity in vivo are thus able to improve anemia in beta-thalassemia and are valuable therapeutic agents for treating iron overload patients or those at risk of iron overload.

[0496] In vivo assessments include assessing the increase in hepcidin gene mRNA transcription in hepatocytes in wild type mice and the decrease in serum iron. The readout of this assessment can be obtained within 24 hours of dosing, allowing rapid confirmation of antibody activity.

[0497] The desired mechanism of action is the inhibition of WT MTP-2 enzyme activity and inhibition of cleavage of downstream substrates on the cell surface of hepatocytes. The immediate impact is an increase in hepcidin mRNA transcription between 2-8 fold observable within 6 hours of administration. The increase in hepcidin expression leads to inhibition of serum iron and thus transferrin saturation in a similar time frame. With continued inhibition of MTP-2 and restriction of serum iron in healthy animals, mean corpuscular volume (MCV) and red cell distribution (RDW) decrease, which typically begins to manifest after 2 weeks, provided the drug remains present and active.

[0498] Antibodies selected for in vivo testing were selected based on their concentration-dependent inhibition of enzymatic activity exhibited against human and mouse MTP-2 protein and cell surface expressed MTP-2. Antibodies were excluded if they were deemed to have potential challenges in expression and / or purification yield, posed development risk, if complete enzyme inhibition was not observed against human or mouse ECD protein in vitro, or if the IC50 value generated was deemed too low.

[0499] Two antibodies that showed weak inhibition in enzyme assays were also tested in vivo. Both performed very poorly in vivo, supporting the hypothesis that antibodies must have a certain level of inhibition of MTP-2 enzymatic activity in order to perform well in vivo. Thus, in vitro inhibition appears to be a necessary criterion for in vivo efficacy. However, this can not always be sufficient to achieve in vivo effect. For some antibodies, we observed strong in vivo performance in the short term, but it did not hold over time. Other factors, such as those related to pharmacokinetics or anti-drug antibodies produced in mice, can impact long-term in vivo performance in these models.

[0500] Example 10. Protocol to determine the effect of anti-MTP-2 antibodies on hepcidin mRNA and serum iron in wild type mice

[0501] Iron assay protocol

[0502] To generate Figure 13 , 14 , 15, 16, 17, and 18, iron quantification was performed using the QuantiChrom TM Iron Assay Kit (Bioassay System, DIFE-250) according to the manufacturer’s protocol.

[0503] Briefly, iron standards were prepared according to the kit protocol. Thereafter, 25 μΐ of standard or sample was added to wells on a 96-well plate, followed by the addition of 200 μΐ of reagent A. The plate was then read on a microplate spectrophotometer at 595 nm (reading A). Ten μΐ of reagent B was then added to the wells, followed by the addition of 10 μΐ of reagent C. The plate was then incubated at room temperature for 40 minutes, and then read on a plate reader at 595 nm (reading B). For all wells, the increase in absorbance was calculated by subtracting reading A from reading B. A standard curve was then plotted, and unknown sample iron values were read from the standard curve.

[0504] To generate Figure 9 , 10 , 11, 12, and 19, serum iron content analysis was performed using the Férène Direct Method Assay Kit according to the protocol provided in the kit.

[0505] Briefly, 25 μΐ of standard or serum from study animals was incubated with 125 μΐ of solution R1 and 25 μΐ of solution R2 in a 96 well, then the plate was read on an envision microplate reader following the 600 nm CLF protocol (absorbance Al). 2.5 μΐ of chromogen was added. After incubation for 20 minutes at room temperature, the plate was read following the same envision protocol as previously (absorbance A2). Results were calculated as follows:

[0506]

[0507]

[0508] Any value calculated as below 0 will be reported as 0.

[0509] Transferrin saturation (TSAT) calculation

[0510] TSAT analysis was performed using the Iron Fixation Latent Period Measurement Kit according to the protocol provided in the kit. Briefly, 25 μΐ of standard or serum from study animals was incubated with 125 μΐ of solution R1 in a 96 well plate for 3 min at room temperature. The plate was then read on an envision microplate reader following the 600 nm CLF protocol (absorbance Al). 25 μΐ of standard or serum from study animals was incubated with 125 μΐ of working solution (50:1 volume of R1 :R2) for 5 min at room temperature. The plate was then read following the same envision protocol as previously (absorbance A2). Results were calculated as follows:

[0511]

[0512] Any value calculated as below 0 will be reported as 0.

[0513] Serum antibody level analysis

[0514] Serum antibody concentrations were determined using the following assay: 96 well plates were coated with 50 μL / well of mouse anti-human IgG4 Fc (2 μg / mL PBS) overnight at 4°C. Plates were washed three times with 300 μL / well of PBD-T (PBS plus 0.1% Tween) using a plate washer. Plates were blocked with 150 μL / well PBS plus 1% BSA for 1 hour at room temperature. Samples were diluted (using pooled mouse sera), QCs and standard curve (10 points (7.81-2000 ng / ml)) were prepared. Plates were then washed three times with 300 μL / well of PBD-T (PBS plus 0.1% Tween) using a plate washer. 50 μL / well of standard curve, samples or QCs were added to the assay plate which was then incubated at room temperature for 1 hour with shaking at 300 RPM. Plates were then washed three times with 300 μL / well of PBD-T (PBS plus 0.1% Tween) using a plate washer. 50 μL / well of HRP conjugated mouse anti-human kappa diluted 1 : 12000 in PBS plus 1% BSA was added to the plate which was then incubated for 1 hour at room temperature with shaking at 300 RPM. Plates were then washed three times with 300 μL / well of PBD-T (PBS plus 0.1% Tween) using a plate washer. 100 μL TMB substrate was added to each well. Incubate at room temperature for 10 minutes protected from light. 100 μL / well of stop solution (1 M sulfuric acid) was then added. The optical density of each well was measured using a microplate reader set to 450 nm, the reference reading was taken at higher than 540 nm. The reference reading was subtracted from the 450 nm reading. The data was then imported into Softmax Pro and the sample concentrations were read off the standard curve using a 4PL curve fit with the weighted factor of the standard being 1 / y regressed.

[0515] RNA extraction

[0516] RNA was prepared from liver samples using Qiagen RNeasy Plus Mini kit according to the protocol in the kit. Frozen samples were thawed on wet ice. Then 600 μΐ Buffer RLT Plus was added to the samples. The samples were then homogenized using a plastic pestle. The samples were then trituated with a 1 ml syringe and 20G needle. The samples were then centrifuged and the supernatant was placed in a gDNA elimination spin column in a 2 ml Eppendorf and the pellet was discarded. The spin column and tube were centrifuged, keeping the flow through and discarding the column. Then 600 μΐ of 70% ethanol was added to the flow through. Then 700 μΐ of sample was placed on a RNeasy spin column in a 2 ml collection tube and then centrifuged. The flow through was discarded. The spin column was then placed back in a 2 ml collection tube. And 700 μΐ of Buffer RW1 was added to the spin column. The spin column was then centrifuged and the liquid flow through was discarded. The spin column was placed back into a 2 ml collection tube and 500 μΐ of Buffer RPE was added to the spin column and the spin column was centrifuged. The flow through was discarded and the column was washed with Buffer RPE as above. Centrifuged at 8000 x g for 15 seconds. The spin column was placed into a new 2 ml collection tube and centrifuged for 1 minute to dry the membrane. The spin column was placed in a new 1.5 ml collection tube and 30-50 μΐ of RNase-free water was added directly to the spin column membrane. The spin column was centrifuged for 1 minute to elute the RNA. The level of RNA in the flow through was estimated using a nanodrop (see below) and the solution was stored at -20°C or -80°C.

[0517] qPCR analysis

[0518] Following mRNA extraction, the mRNA from the liver of each mouse was quantified by nanodrop and all were normalized to 5 ng / μΐ. Briefly, the transcriptional level of mouse hepcidin (hamp) mRNA was measured by qRT-PCR and normalized to the mouse hypoxanthine-guanine phosphoribosyltransferase (HPRT) mRNA housekeeping gene. Then 5 μΐ of mRNA extract (25 ng total) was mixed with 10 μΐ of QuantiTect probe RT-PCR kit, mixed with 1 μΐ of 20X hamp FAM probe mix, 1 μΐ of 20X HPRT VIC probe mix, 0.5 μΐ of 40X taqman RT enzyme mix, 2.5 μΐ of RNA-free H2O in a 96 well half skirt qRT-PCR plate in a 20 μΐ final volume. The setup of the qRT-PCR reaction included a 15 minute reverse transcription step at 48°C followed by a 10 minute activation step at 95°C and then 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Then the -ΔCt value was calculated by subtracting the Ct value of Hamp from HPRT.

[0519] Example 11. Single dose evaluation of four IgG / lambda anti-MTP2 antibodies for reduction of serum iron and transferrin saturation at 24h in normal mice Figure 9

[0520] NORI-009, NORI-010, NORI-012 and NORI-034 were included in the first evaluation in healthy mice. Nine-week-old C57BL / 6 male mice received one intraperitoneal injection of 10 mg / kg (150 μl / mouse) of antibody, 5 mice per group. Mice were sacrificed 24 h after injection. As a positive control for hepcidin induction, three nine-week-old C57BL / 6 male mice were injected with LPS (1 μg / g body weight) and sacrificed 4 hours later. LPS (lipopolysaccharide from E. coli) mimics a bacterial infection and therefore the mice respond by inducing hepcidin expression to decrease serum iron. LPS causes an acute inflammatory response and acts as a positive control for a drug known to cause the maximum hepcidin increase based on the inflammatory response. Real-time PCR of hamp, Idl, Atho8, SMAD7, CRP and Saa3 mRNA was performed on liver tissue. Hematological parameters were also determined. Red blood cell count was determined and hemoglobin was measured. Serum iron was determined in all mice and transferrin saturation was calculated.

[0521] NORI-034 was not seen to increase hepcidin expression above the levels seen in the isotype control treated mice. On the other hand, NORI-009, NORI-010 and NORI-012 all increased hepcidin expression above the mean of the isotype control treated group by at least 1 ct value 24 hours after dosing and equal to the value induced by the LPS positive control 4 hours after dosing. The result was a decrease in serum iron content in the mice treated with these antibodies to a mean concentration of less than 40 μg / dl. Example 12. Time course evaluation of NORI-010 after a single intraperitoneal injection in normal mice .

[0522] This experiment confirmed the biological relevance of MTP-2 in the BMP / SMAD / hepcidin pathway and for the first time showed that serum iron can be decreased in normal mice by directed inhibition with antibodies. Since NORI-034 was not active in vivo, this antibody was not further investigated.

[0523] Figure 10

[0524] Antibody NORI-010 is one of the antibodies that showed preferred characteristics in Example 11, and it was of interest to determine the effects observed over time. C57BL / 6 male mice received one intraperitoneal injection of 10 mg / kg (150 μl / mouse) of antibody, 5 mice per group. Groups were terminated at 24 h, 72 h, 1 week and 2 weeks, 5 mice per group. Analysis was the same as in Example 11, but also included hematocrit (HCT), mean corpuscular hemoglobin (MCH) and red cell distribution width (RDW), measures of cell diameter related to red cell volume.

[0525] After administration of 10 mg / kg antibody, hepcidin elevation was maintained for 2 weeks, during which serum iron and TSAT were decreased. MCV values were decreased due to consistent iron restriction. Figure 11 .

[0526] After administration of 3 mg / kg antibody, the effect on hepcidin elevation was lost at 2 weeks due to the lower dose. Nonetheless, serum iron and TSAT remained decreased over 2 weeks, and MCV was also decreased as with the 10 mg / kg dose. Example 13. Evaluation of two IgG / kappa and two IgG / lambda anti-MTP2 antibodies for reduction of serum iron and transferrin saturation at 24h in normal mice .

[0527] Figure 12 Example 14. Time course evaluation of NORI-008 after a single intraperitoneal injection at 10, 3 and 1 mg / kg in normal mice

[0528] NORI-008 in the mouse IgGl (moIgGl) format was evaluated for in vivo activity. NORI-010 huIgG4PE was included as a positive control antibody for in vivo anti-MTP-2 activity. NORI-036 and NORI-037 in the moIgGl and huIgG4PE formats, respectively, were included as negative control antibodies. The latter control antibodies are cross-reactive binders to MTP-2, but do not inhibit the enzymatic activity of human or mouse ECD protein in vitro.

[0529] 9 week old C57BL / 6 male mice received one intraperitoneal injection of 10 mg / kg (150 μl / mouse) of antibody, 3-5 mice per group. Mice were sacrificed 24 h after injection. Real-time PCR of hamp, Idl, Atho8, SMAD7, CRP and Saa3 mRNA was performed on liver tissue. Hematological parameters were also determined. Red blood cell counts were determined, and hemoglobin was measured. Serum iron was determined for all mice and transferrin saturation was calculated.

[0530] Here we found that NORI-008, which has very strong affinity for human and mouse MTP-2, is also active in vivo. It was found to have the same activity as NORI-010 in increasing hepcidin RNA expression and decreasing serum iron content at the 24 hour time point. Figure 13 .

[0531] This demonstrates that both antibodies maximally inhibit MTP-2 activity in vivo. NORI-036 and NORI-037 are binders to MTP-2, but have no effect on hepcidin expression or serum iron in vivo. This demonstrates that the in vitro screening strategy successfully generated antibodies that can successfully block MTP-2 in vivo to induce a biological response.

[0532] Example 15. In vivo evaluation of six IgG4 / kappa anti-MTP2 antibodies for reduction of serum iron and transferrin saturation at 1 week time point in normal mice

[0533] This experiment shows the dose-time relationship following intraperitoneal injection of anti-MTP-2 antibody NORI-008 on serum iron concentration.

[0534] Male C57BL6J mice (22-28 g, n=5 / group) were given a single intraperitoneal injection of the fully human IgG4 anti-MTP-2 antibody NORI-008 at 2, 20 or 200 μg. One group received a 200 μg dose of a human IgG4 isotype control antibody and was picked on day 1. An additional 9 groups consisted of 3 groups per dose of NORI-008 (2, 20 or 200 μg), with each group picked at a separate dose. One group of animals treated with each dose was bled on days 1, 7 and 14 and serum iron concentration was determined to assess efficacy of NORI-008.

[0535] Samples from each dose and time point were then analyzed for hepcidin expression in the liver and antibody levels in the serum (PK analysis) on day 7. No antibody was measured in the 2 μg group at any time point, and only three of five animals had measurable levels 24 hours after the 20 μg dose.

[0536] At the 200 μg dose, the effect of NORI-008 lasted at least 1 week in vivo, after which hepcidin expression and serum iron returned to normal at the 2 week time point. At the 20 μg dose, the effect lasted at least 1 day, after which it returned to normal at the 1 week time point. No effect was observed with the 2 μg dose. The duration of effect correlated with the PK values for antibody concentration. That is, when the antibody was below a critical concentration, the effect of MTP-2 inhibition was lost and hepcidin expression and serum iron returned to normal. Figure 14 .

[0537] Example 16. In vivo evaluation of two IgG / kappa and one IgG / lambda anti-MTP2 antibodies in huIgG4 and moIgGl backbone for reduction of serum iron at 1 week time point (10 mg / kg intraperitoneal dose) Figure 15

[0538] This study evaluated the ability of six fully human IgG4 anti-MTP-2 antibodies to reduce serum iron concentration following intraperitoneal injection.

[0539] Male C57BL6J mice (25-32 g, n=3-5 / group) were given a single 10 mg / kg intraperitoneal injection of fully human IgG4 anti-MTP-2 antibodies. Eight groups were included in the study, each animal receiving a single 10 mg / kg intraperitoneal dose of one of seven fully human IgG4 PE anti-MTP2 antibodies NORI-005, NORI-004, NORI-002, NORI-001, NORI-007, NORI-006, and NORi-010 or a human IgG4 isotype control antibody. NORI-010 was added as a positive control and benchmark anti-MTP-2 antibody for comparison with the other antibodies used in this experiment. All groups contained 5 animals except for NORI-005 (n=3) and NORI-002 (n=4). Animals were bled on day 7 and serum iron concentration was determined to assess iron parameters, hamp mRNA, and IgG levels for the anti-MTP-2 antibodies.

[0540] All tested antibodies showed an increase in hepcidin mRNA expression, and thus a corresponding decrease in serum iron levels. Samples from all tested groups were then analyzed for antibody levels in serum (PK analysis) on day 7, and showed different levels of antibody exposure at this time point and dose. Example 17. In vivo evaluation of NORI-010 after a single intraperitoneal (ip) or subcutaneous (sc) injection in normal mice, read out 7 days post injection .

[0541] Figure 16 Example 18. In vivo evaluation of the dose / time response of anti-MTP-2 antibodies NORI-008 and NORI-010 after a single intraperitoneal injection in normal rats

[0542] This study evaluated the ability of three anti-MTP-2 antibodies in fully human IgG4 or mouse IgGl format to decrease serum iron concentration after subcutaneous injection.

[0543] Male C57BL6J mice (25-32 g, n=5 / group) were given a single 10 mg / kg subcutaneous injection of fully human IgG4 or molgG1 anti-MTP-2 antibodies. Seven groups were included in the study, using one of three anti-MTP2 antibodies NORI-011, NORI-003, and NORI-006 as either human IgG4 or murine IgGl, or a human IgG4 isotype control antibody. Animals were bled on day 7 and serum iron concentration was determined to assess efficacy of the anti-MTP-2 antibodies.

[0544] Figure 17 .

[0545] Example 19. In vivo evaluation of the dose / time response of anti-MTP-2 antibodies NORI-003, NORI-006, NORI-008 and NORI-010 after a single subcutaneous injection in normal rats Example 20. In vivo evaluation in a mouse model of beta thalassemia after a single intraperitoneal injection of NORI-010

[0546] This experiment was used to evaluate whether fully human IgG4 anti-MTP-2 antibody NORI-010 produced a similar decrease in serum iron concentration after a 10 mg / kg subcutaneous injection as seen with a 10 mg / kg intraperitoneal dose.

[0547] Male C57BL6J mice (23-29 g, n=4-5 / group) were given a single 10 mg / kg subcutaneous or intraperitoneal injection of fully human IgG4 anti-MTP-2 antibody. Two groups received a single 10 mg / kg intraperitoneal dose or a single 10 mg / kg subcutaneous dose of NORI-010 (n=5 per group). Two additional groups received a 10 mg / kg dose of human IgG4 isotype control antibody intraperitoneally or subcutaneously.

[0548] Animals were bled on day 7 and serum iron concentration and IgG levels were determined. Figure 19 .

[0549] The results of this experiment show that for both intraperitoneal and subcutaneous administration methods, the concentration of antibody in serum at the 1 week time point was similar. As a result, the reduction in serum iron was also similar for both administration methods, and as seen previously, was reduced by MTP-2 inhibition.

[0550] Example 21. In vivo evaluation of anti-MTP-2 antibodies NORI-003, NORI-006, NORI-008 and NORI-010 in a mouse model of beta thalassemia with and without co-treatment with erythropoietin ​

[0551] This experiment was used to evaluate the dose-time relationship of two fully human IgG4 anti-MTP-2 antibodies following intraperitoneal injection.

[0552] Male Wistar rats (260-320 g, n=3 / group) were given a single 3 or 10 mg / kg intraperitoneal injection of fully human IgG4 anti-MTP-2 antibodies NORI-008 and NORI-010. A hIgG4 isotype control antibody was given as a negative control by intraperitoneal injection at 10 mg / kg (labeled "isotype").

[0553] Animals were bled on days 1, 3, 7, 9, 14 and 21. Serum iron and serum antibody concentrations were measured at each time point.

[0554] NORI-010 at 10 mg / kg produced a pharmacological effect and reduced serum iron for up to 9 days, after which it returned to normal, while the effect of 3 mg / kg lasted only 24 hours. NORI-008 at 10 mg / kg produced an effect and reduced serum iron for up to 9 days, after which it returned to normal, while the effect of 3 mg / kg lasted only 72 hours. The PD of these antibodies was closely related to IgG serum levels, and when antibody concentration decreased, the iron-reducing effect was lost. ​ .

[0555] ​ ​

[0556] In vivo evaluation of the dose / time response following a single subcutaneous injection of anti-MTP-2 antibodies in normal rats.

[0557] Male Wistar rats (260-320 g, n=5 / group) were given a single 10 mg / kg s.c. injection of the fully human IgG4 anti-MTP-2 antibodies NORI-003, NORI-006, NORI-008 and NORI-010. A huIgG4 isotype control antibody (labeled "isotype") was dosed at 10 mg / kg as a negative control.

[0558] Animals were bled on days 1, 3, 7, 9, 14 and 21. Serum iron and serum antibody concentrations were measured at each time point.

[0559] NORI-008 and NORI-010 again showed a similar duration of action to Example 18, with the effect on serum iron lasting until day 9, after which it returned to normal. NORI-003 and NORI-006 both showed a more favorable duration of action, with serum iron remaining suppressed throughout the 21 -day study.

[0560] The PK analysis of this study revealed a key difference in Cmaxfor the four antibodies, with NORI-003 and NORI-006 having a much higher serum antibody concentration on day 3 compared to NORI-008 and NORI-010. As a result, these two antibodies showed a more favorable PK profile and remained above the critical serum antibody concentration in order to maintain suppression of MTP-2 for a longer period of time. As a result, the effect on serum iron was maintained. Figure 18.

[0561] ​

[0562] The selected antibody NORI-010 was evaluated in a hybrid mouse model of intermediate beta thalassemia (Hbbth3 / +) that is heterozygous for the deletion of the b1 and b2 globin genes. Hbbth3 / + mice exhibit similar features to human intermediate beta- thalassemia, including Hb levels between 7 and 9 g / dL, abnormal red blood cell morphology, increased reticulocyte count, ineffective and extramedullary erythropoiesis, hepatosplenomegaly and liver and spleen iron overload, which is a complex phenotype that worsens with age. Following the activity of NORI-010 observed in Examples 18-20, the antibody was injected intraperitoneally at 10 mg / kg and animals were terminated 2 weeks after this single dose (n=3 per time point). In addition, a group of animals was analyzed for hepcidin mRNA 24 h later. A human IgG4 isotype control antibody was used at the same dose as a negative control.

[0563] The results of the following parameters were recorded:

[0564] - hepcidin mRNA levels in the liver (24 h and 2 weeks, according to Method Example 10)

[0565] - Id1 mRNA levels in the liver (24h and 2 weeks, according to method example 10)

[0566] - Serum iron concentration (2 weeks, according to method example 10)

[0567] - Calculated transferrin saturation (2 weeks, according to method example 10)

[0568] - Mean corpuscular volume (2 weeks)

[0569] The results show that after administration of NORI-010, at 24h, the hepcidin levels were already higher than with the isotype control. This difference was maintained over a period of two weeks. In agreement, the serum iron levels were reduced by about 52% compared to the isotype control and the calculated transferrin saturation was reduced by 47% at the two-week time point compared to animals treated with the isotype control. ​ .

[0570] ​ 8-week repeat-dose evaluation of NORI-011-M treatment

[0571] The aim of this study was to observe the impact on hematological parameters after a longer treatment period than example 20. To this end, Hbbth3 / + mice (n=5 / group) were injected intraperitoneally once a week with 10 mg / kg of NORI-011-M antibody for 8 weeks. For this long-term repeated dose experiment, the isotype of NORI-011 was reformatted with murine constant regions (murine IgG1 and murine lambda constant regions) to avoid or reduce the immunogenicity and production of anti-MTP2 antibodies in mice. Regarding this reformulation, also the NORI-011 VH domain C-terminal sequence was slightly modified and the modified antibody was renamed NORI-011-M. The sequence of NORI-011-M is shown in Table S. The control used here is the mouse isotype IgG1.

[0572] Furthermore, the study tried to examine the impact of co-administration of erythropoietin (epo), which is known to improve some key parameters, but has a detrimental impact on iron loading and spleen size due to excessive cell death. The positive effects of this combination have been observed in experiments by providing epo through overexpression of recombinant fibroblasts and by reducing the proteolysis-2 activity in Hbbth3 / + mice using Tmprss6 antisense oligonucleotide treatment (paper #164 60th American Society of Hematology Annual Meeting 2018). In this study, clinical grade epo (darbepoetin alfa) was co-administered with NORI-011-M at 30 pg / kg once weekly in one group of the study. As a control, epo was also co-administered with isotype control antibody, which represents the impact of epo alone on the study readouts. Figure 20 shows the results for individual hematological parameters and hepcidin mRNA levels in liver samples.

[0573] The results show that hepcidin levels can be kept at elevated levels throughout the 8-week experimental course (a). Thus, it can be expected that this will lead to iron restriction and subsequently a decrease in serum iron, liver iron content (b) and MCV (f) as seen in the above described examples. The red blood cell count and hemoglobin are not substantially increased with NORI-011-M alone. However, the maturation of red blood cells in the spleen is improved with a much larger proportion of cells in stage V (mature) compared to any other group (i). The administration of NORI-011-M alone also reduces the increase in spleen weight by about half, which indicates a normalizing effect of the iron supply restriction caused by the blockade of NORI-011-M on the maturation of red blood cells (k).

[0574] The use of epo (in combination with isotype control) has a positive impact on red blood cell count (c) and hemoglobin (d) as expected for a stimulator of erythropoiesis. However, as previously known, this increased erythropoiesis leads to an increase in cell death and associated increase in spleen size (index) in the context of a beta globin synthesis deficiency (k) and no improvement in the maturation to functional and high quality red blood cells (i). As expected, epo does not increase hepcidin levels and thus has no impact on liver iron levels (c).

[0575] The combination treatment of NORI-011-M and epo leads to a combination of positive effects. This manifests in a slightly reduced impact on red blood cell count (c) and hemoglobin (d) compared to epo alone, but overall a more balanced therapeutic effect including a continued reduction of liver iron overload (b) and reduction of splenomegaly (k) compared to epo alone. These cooperative effects are considered to be optimizable in a clinical setting by optimizing the therapeutic ratio of proteolysis inhibition and epo stimulation.

[0576] Example 22. A mouse model of beta thalassemia with and without co-treatment of ActRIIB-Fc fusion protein8-week repeat-dose evaluation of NORI-011-M treatment in the presence of ActRIIB-Fc fusion protein

[0577] In this study, we demonstrate that co-treatment with NORI-11-M and Activin Receptor IIB Fc fusion protein provides a beneficial therapeutic effect compared to treatment with NORI-11-M alone and ActRIIB-Fc alone. This synergy can reflect the different modes of action of the two agents, where ActRIIB-Fc promotes maturation of red cell precursors in the process of erythropoiesis, while NORI-11-M causes iron restriction and normalization, thus slowing down erythropoiesis, leading to a more efficient production of more mature red cells.

[0578] The work in this study was performed using the methods and procedures generally as described above in Example 21 with multiple administrations of the two agents over 8 weeks. As before, Hbbth3 / + mice (n=5 / group) were injected intraperitoneally with 10 mg / kg NORI-011-M antibody once a week for 8 weeks. ActRIIB-Fc was injected intraperitoneally twice a week at 10 mg / kg for 8 weeks. The frequency of administration of ActRIIb-Fc was doubled compared to the antibody, which reflects the administration schedule of a similar activin receptor ligand trap molecule published previously (Suragani RN, Cawley SM, Li R, et al. Modified activin receptor IIB ligand trap mitigates ineffective erythropoiesis and disease complications in murine β-thalassemia. Blood. 2014;123(25):3864-3872. doi: 10.1182 / blood-2013-06-511238 and Dussiot et al. 2014) for ActRIIA-Fc.

[0579] The ActRIIB-Fc construct used in this example is a modified human ActRIIB extracellular domain (uniprot-Q13705, residues 26-131) with a L79D modification, fused to a mouse IgG2a-Fc domain (uniprot-P01863, residues 99-330) via a short 3x glycine linker, expressed in suspension CHO cells and purified via the Fc domain.

[0580] Treatment groups were compared to healthy, untreated wild-type mice (“WT”) and Hbbth3 / + mice treated with a mouse IgG1 control (“MoIgG1”).

[0581] Figure 21 shows the results for individual hematological parameters and hepcidin mRNA levels in liver samples. In line with Example 21 above, the results again indicate that the use of NORI-11-M keeps hepcidin levels at elevated levels throughout the 8-week course (Figure 21a), demonstrating that NORI-11-M has an effect to increase hepcidin expression levels, leading to reduced liver iron levels (Figure 21b), reduced serum iron, and reduced mean corpuscular volume (MCV) (Figure 21f). No increase in hepcidin (hamp) mRNA was observed for treatment with ActRIIB-Fc alone, and co-treatment was identical to NORI-11-M alone. Thus, ActRIIB-Fc was not able to directly increase hepcidin expression or, correspondingly, reduce liver iron levels, despite the higher dosing frequency.

[0582] Red blood cell mean corpuscular hemoglobin (MCH) was reduced in mice treated with NORI-11-M (which reflects a limitation in iron supply) and also by ActRIIB-Fc (Figure 21h), which reflects the production of a greater number of cells (shown by the increase in total red blood cell count and hemoglobin) and thus less hemoglobin available to each cell. Mean corpuscular volume (MCV) was also reduced (Figure 21f). Generally, low MCV indicates microcytic anemia, but in this case, the iron limitation (caused by the antibody) or hemoglobin deficiency (for ActRIIB-Fc, caused by increased maturation of more RBCs) leads to reduced availability per cell, as the MCH results have already indicated.

[0583] All treatment groups improved the "quality" of the red blood cells produced, as reflected by the decrease (normalization) of the red blood cell distribution width (RDW) (Figure 21g). The homogeneity of the red blood cells was thus improved. The use of NORI-011-M alone slightly increased the red blood cell count (Figure 21c), but neither the hemoglobin (Figure 21d) nor the hematocrit (Figure 21e) increased. ActRIIB-Fc had a large effect on all these parameters. The combination did not lead to a further increase compared to the treatment with ActRIIB-Fc alone, indicating that the maturation effect on red blood cell production was driven by ActRIIB-Fc and not by NORI-11-M. It is noteworthy, however, that NORI-11-M did not counteract the effect of ActRIIB-Fc. With treatment by ActRIIB-Fc alone or in combination with NORI-11-M, the red blood cell count returned to wild-type levels, and both the hematocrit and the hemoglobin levels increased significantly. This is important because it indicates that the iron reduction caused by NORI-11-M did not reverse the beneficial effect on red blood cell production generated by ActRIIB-Fc. Thus, overall, when the two treatments are used in combination, the advantages of both treatments can still be realized, to reduce iron overload and increase the production / maturation of red blood cells.

[0584] Furthermore, the use of the two agents together actually results in a greater overall benefit, with a therapeutic potential that exceeds that of their use alone, as seen, for example, by the effect of reducing splenomegaly.

[0585] The maturation of red blood cells in the spleen was improved, with a much greater proportion of cells in stage V (mature) compared to any other group (Figure 21i). The increase in spleen weight was reduced with both treatments compared to the untreated control, indicating that even the limitation of iron supply caused by NORI-011-M has a normalizing effect on red blood cell maturation efficiency (Figure 21k).

[0586] We reported in Example 21 that the use of EPO had a positive effect on the red blood cell count (Figure 20c) and on the total hemoglobin (Figure 20d), as expected for a stimulator of red blood cell production. However, as is known in the medical field, this increase in stimulation of red blood cell production also causes an increase in cell death and apoptosis and a further increase in the size and weight of the spleen (so-called splenomegaly) in the case of a persistent deficiency in the synthesis of beta globin (Figure 20k). Here, in this example, the use of NORI-11-M and ActRIIB-Fc both did not lead to an increase in the weight of the spleen compared to the untreated animals (Figure 21k). Indeed, the weight of the spleen was actually significantly reduced, although not to the level of healthy animals. The combined treatment of NORI-011-M and ActRIIB-Fc appeared even better than the single therapies alone by even further reducing the weight of the spleen.

[0587]

[0588]

[0589]

[0590]

[0591]

[0592]

[0593]

[0594]

[0595]

[0596]

[0597]

[0598]

[0599]

[0600]

[0601]

[0602]

[0603]

[0604]

[0605]

[0606]

[0607]

[0608]

[0609]

[0610]

[0611]

[0612]

[0613]

[0614]

[0615]

[0616]

[0617]

[0618]

[0619]

[0620]

[0621]

[0622]

[0623]

[0624]

[0625]

[0626]

[0627]

[0628]

[0629]

[0630]

[0631]

[0632]

[0633]

[0634]

[0635]

[0636]

[0637]

[0638]

[0639]

[0640]

[0641]

[0642]

[0643]

[0644]

[0645]

[0646]

[0647]

[0648]

[0649]

[0650]

[0651]

[0652]

[0653]

[0654]

[0655]

[0656]

[0657]

[0658]

[0659]

[0660]

[0661]

[0662]

[0663]

[0664]

[0665]

[0666]

[0667]

[0668]

[0669]

[0670]

[0671]

[0672]

[0673]

[0674]

[0675]

[0676]

[0677]

[0678]

[0679]

[0680]

[0681]

[0682]

[0683]

[0684]

[0685] SEQUENCE LISTING <110> Kymab Limited <120> Treatment of physiologic iron overload <130> K00066-1 WO <150> GB1917524.9 <151> 29-NOV-2019 <150> GB1917882.1 <151> 06-DEC-2019 <160> 580 <170> PatentIn 3.5 <210> 1 <211> 855 <212> PRT <213> Homo sapiens <400> 1 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 Gin Ala Arg Val Val Gly Gly Thr Asp Ala Asp Glu Gly 610 615 620 Glu Trp Pro Trp Gin Val Ser Leu His Ala Leu Gly Gin Gly His Ile 625 630 635 640 Cys Gly Ala Ser Leu Ile Ser Pro Asn Trp Leu Val Ser Ala Ala His 645 650 655 Cys Tyr Ile Asp Asp Arg Gly Phe Arg Tyr Ser Asp Pro Thr Gin Trp 660 665 670 Thr Ala Phe Leu Gly Leu His Asp Gin Ser Gin Arg Ser Ala Pro Gly 675 680 685 Val Gin Glu Arg Arg Leu Lys Arg Ile Ile Ser His Pro Phe Phe Asn 690 695 700 Asp Phe Thr Phe Asp Tyr Asp Ile Ala Leu Leu Glu Leu Glu Lys Pro 705 710 715 720 Ala Glu Tyr Ser Ser Met Val Arg Pro Ile Cys Leu Pro Asp Ala Ser 725 730 735 His Val Phe Pro Ala Gly Lys Ala Ile Trp Val Thr Gly Trp Gly His 740 745 750 Thr Gln Tyr Gly Gly Thr Gly Ala Leu Ile Leu Gln Lys Gly Glu Ile 755 760 765 Arg Val Ile Asn Gln Thr Thr Cys Glu Asn Leu Leu Pro Gln Gln Ile 770 775 780 Thr Pro Arg Met Met Cys Val Gly Phe Leu Ser Gly Gly Val Asp Ser 785 790 795 800 Cys Gln Gly Asp Ser Gly Gly Pro Leu Ser Ser Val Glu Ala Asp Gly 8​​​​​​​​​​​​​​​​​​​​​​​​​​​Gln Val Ala Gly Gly Gin Gly Asp Gly Gly Asp Gly Glu Glu Ala Glu 20 25 30 Pro Glu Gly Met Phe Lys Ala Cys Glu Asp Ser Lys Arg Lys Ala Arg 35 40 45 Gly Tyr Leu Arg Leu Val Pro Leu Phe Val Leu Leu Ala Leu Leu Val 50 55 60 Leu Ala Ser Ala Gly Val Leu Leu Trp Tyr Phe Leu Gly Tyr Lys Ala 65 70 75 80 Glu Val Met Val Ser Gin Val Tyr Ser Gly Ser Leu Arg Val Leu Asn 85 90 95 Arg His Phe Ser Gin Asp Leu Thr Arg Arg Glu Ser Ser Ala Phe Arg 100 105 110 Ser Glu Thr Ala Lys Ala Gin Lys Met Leu Lys Glu Leu He Thr Ser 115 120 125 Thr Arg Leu Gly Thr Tyr Tyr Asn Ser Ser Ser Val Tyr Ser Phe Gly 130 135 140 Glu Gly Pro Leu Thr Cys Phe Phe Trp Phe He Leu Gin He Pro Glu 145 150 155 160 His Arg Arg Leu Met Leu Ser Pro Glu Val Val Gin Ala Leu Leu Val 165 170 175 Glu Glu Leu Leu Ser Thr Val Asn Ser Ser Ala Ala Val Pro Tyr Arg 180 185 190 Ala Glu Tyr Glu Val Asp Pro Glu Gly Leu Val Ile Leu Glu Ala Ser 195 200 205 Val Lys Asp Ile Ala Ala Leu Asn Ser Thr Leu Gly Cys Tyr Arg Tyr 210 215 220 Ser Tyr Val Gly Gln Gly Gln Val Leu Arg Leu Lys Gly Pro Asp His 225 230 235 240 Leu Ala Ser Ser Cys Leu Trp His Leu Gln Gly Pro Lys Asp Leu Met 245 250 255 Leu Lys Leu Arg Leu Glu Trp Thr Leu Ala Glu Cys Arg Asp Arg Leu 260 265 270 Ala Met Tyr Asp Val Ala Gly Pro Leu Glu Lys Arg Leu Ile Thr Ser 275 280 285 Val Tyr Gly Cys Ser Arg Gln Glu Pro Val Val Glu Val Leu Ala Ser 290 295 300 Gly Ala Ile Met Ala Val Val Trp Lys Lys Gly Leu His Ser Tyr Tyr 305 310 315 320 Asp Pro Phe Val Leu Ser Val Gln Pro Val Val Phe Gln Ala Cys Glu 325 330 335 Val Asn Leu Thr Leu Asp Asn Arg Leu Asp Ser Gin Gly Val Leu Ser 340 345 350 Thr Pro Tyr Phe Pro Ser Tyr Tyr Ser Pro Gin 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 Gin Lys Tyr Asp Leu Pro Cys Thr Gin Gly 385 390 395 400 Gln Trp Thr Ile Gin Asn Arg Arg Leu Cys Gly Leu Arg Ile Leu Gin 405 410 415 Pro Tyr Ala Glu Arg Ile Pro Val Val Ala Thr Ala Gly Ile Thr Ile 420 425 430 Asn Phe Thr Ser Gin Ile Ser Leu Thr Gly Pro Gly Val Arg Val His 435 440 445 Tyr Gly Leu Tyr Asn Gin 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 Val Lys Asp Cys 465 470 475 480 Pro Asn Gly Leu Asp Glu Arg Asn Cys Val Cys Arg Ala Thr Phe Gin 485 490 495 Cys Lys Glu Asp Ser Thr Cys lie Ser Leu Pro Lys Val Cys Asp Gly 500 505 510 Gln Pro Asp Cys Leu Asn Gly Ser Asp Glu Glu Gin Cys Gin Glu Gly 515 520 525 Val Pro Cys Gly Thr Phe Thr Phe Gin Cys Glu Asp Arg Ser Cys Val 530 535 540 Lys Lys Pro Asn Pro Gin Cys Asp Gly Arg Pro Asp Cys Arg Asp Gly 545 550 555 560 Ser Asp Glu Glu His Cys Asp Cys Gly Leu Gin Gly Pro Ser Ser Arg 565 570 575 Ile Val Gly Gly Ala Val Ser Ser Glu Gly Glu Trp Pro Trp Gin Ala 580 585 590 Ser Leu Gin Val Arg Gly Arg His lie Cys Gly Gly Ala Leu lie Ala 595 600 605 Asp Arg Trp Val lie Thr Ala Ala His Cys Phe Gin Glu Asp Ser Met 610 615 620 Ala Ser Thr Val Leu Trp Thr Val Phe Leu Gly Lys Val Trp Gin Asn 625 630 635 640 Ser Arg Trp Pro Gly Glu Val Ser Phe Lys Val Ser Arg Leu Leu 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 Arg Ser Ala Ala Val Arg Pro Val Cys 675 680 685 Leu Pro Ala Arg Ser His Phe Phe Glu Pro Gly Leu His Cys Trp Ile 690 695 700 Thr Gly Trp Gly Ala Leu Arg Glu Gly Gly Pro Ile Ser Asn Ala Leu 705 710 715 720 Gln Lys Val Asp Val Gln Leu Ile Pro Gln Asp Leu Cys Ser Glu Val 725 730 735 Tyr Arg Tyr Gln Val Thr 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 Lys Ala Leu Ser Gly Arg Trp Phe Leu Ala Gly Leu Val Ser Trp Gly 770 775 780 Leu Gly Cys Gly Arg Pro Asn Tyr Phe Gly Val Tyr Thr Arg Ile Thr 785 790 795 800 Gly Val Ile Ser Trp Ile Gln Gln Val Val Thr 805 810 <210> 3 <211> 843 <212> PRT <213> Homo sapiens <400> 3 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 Lys Lys Lys Val Pro Phe Trp Asn Val Gln Asn Lys 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 Gin 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 Gin Thr Ser Ile 195 200 205 Ile Asn Arg Thr Ser Val Gly Ser Leu Gin 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 Gin Tyr Phe Tyr Ala Gin His Leu 245 250 255 Ser Leu His Tyr Pro Leu Gin 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 Gin Ile Gin 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 Gin 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 Gin Thr Ile 435 440 445 Phe Arg Val Pro Ser Pro Leu Val His lie Gin Leu Gin Cys Ser Ser 450 455 460 Arg Leu Ser Asp Lys Pro Leu Leu Ala Glu Tyr Gly Ser Tyr Asn lie 465 470 475 480 Ser Gin Pro Cys Pro Val Gly Ser Phe Arg Cys Ser Ser Gly Leu Cys 485 490 495 Val Pro Gin Ala Gin Arg Cys Asp Gly Val Asn Asp Cys Phe Asp Glu 500 505 510 Ser Asp Glu Leu Phe Cys Val Ser Pro Gin Pro Ala Cys Asn Thr Ser 515 520 525 Ser Phe Arg Gin His Gly Pro Leu lie Cys Asp Gly Phe Arg Asp Cys 530 535 540 Glu Asn Gly Arg Asp Glu Gin Asn Cys Thr Gin Ser lie Pro Cys Asn 545 550 555 560 Asn Arg Thr Phe Lys Cys Gly Asn Asp lie Cys Phe Arg Lys Gin 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 lie lie Gly 595 600 605 Gly Thr Asp Thr Leu Glu Gly Gly Trp Pro Trp Gin 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 Gin 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 Gin Leu Ser Ile Ala Trp 690 695 700 Pro Glu Thr Leu Lys Gin Leu Ile Gin Pro Ile Cys Ile Pro Pro Thr 705 710 715 720 Gly Gin 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 Gin Gin Ala 740 745 750 Glu Val Glu Leu Ile Asp Gin 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 835 840 <210> 4 <211> 811 <212> PRT <213> Mus Musculus <400> 4 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 Gin Val Leu Pro Leu Lys Gly Pro Asp Gin 225 230 235 240 Gln Thr Thr Ser Cys Leu Trp His Leu Gin 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 Gin 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 Gin Asp Cys Gin 325 330 335 Val Asn Leu Thr Leu Glu Gly Arg Leu Asp Thr Gin 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 Gin Lys Tyr Asn Arg Leu Cys Thr Gin Gly 385 390 395 400 Gln Trp Met lie Gin Asn Arg Arg Leu Cys Gly Phe Arg Thr Leu Gin 405 410 415 Pro Tyr Ala Glu Arg lie Pro Met Val Ala Ser Asp Gly Val Thr lie 420 425 430 Asn Phe Thr Ser Gin lie Ser Leu Thr Gly Pro Gly Val Gin Val Tyr 435 440 445 Tyr Ser Leu Tyr Asn Gin 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 lie Lys Asp Cys 465 470 475 480 Pro Asn Gly Leu Asp Glu Arg Asn Cys Val Cys Arg Ala Met Phe Gin 485 490 495 Cys Gin Glu Asp Ser Thr Cys lie Ser Leu Pro Arg Val Cys Asp Arg 500 505 510 Gln Pro Asp Cys Leu Asn Gly Ser Asp Glu Glu Gin Cys Gin Glu Gly 515 520 525 Val Pro Cys Gly Thr Phe Thr Phe Gin Cys Glu Asp Arg Ser Cys Val 530 535 540 Lys Lys Pro Asn Pro Glu Cys Asp Gly Gin Ser Asp Cys Arg Asp Gly 545 550 555 560 Ser Asp Glu Gin His Cys Asp Cys Gly Leu Gin Gly Leu Ser Ser Arg 565 570 575 Ile Val Gly Gly Thr Val Ser Ser Glu Gly Glu Trp Pro Trp Gin Ala 580 585 590 Ser Leu Gin 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 Gin Gin Asp Ser Met 610 615 620 Ala Ser Pro Lys Leu Trp Thr Val Phe Leu Gly Lys Met Arg Gin 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 Gin Gin 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 Gin His Cys Trp He 690 695 700 Thr Gly Trp Gly Ala Gin Arg Glu Gly Gly Pro Val Ser Asn Thr Leu 705 710 715 720 Gln Lys Val Asp Val Gin Leu Val Pro Gin Asp Leu Cys Ser Glu Ala 725 730 735 Tyr Arg Tyr Gin Val Ser Pro Arg Met Leu Cys Ala Gly Tyr Arg Lys 740 745 750 Gly Lys Lys Asp Ala Cys Gin 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 He Asn Trp He Gin Gin Val Leu Thr 805 810 <210> 5 <211> 811 <212> PRT <213> Rattus norvegicus <400> 5 Met Pro Arg Cys Phe Gin Leu Pro Cys Ser Thr Arg Met Pro Thr Ala 1 5 10 15 Glu Val Pro Gin Ala Ala Gly Gly Gin Gly Asp Gly Gly Asp Gly Glu 20 25 30 Glu Ala Ala Glu Pro Glu Gly Val Phe Lys Ala Pro Arg Asn Ala Lys 35 40 45 Arg Lys Asp Arg Asp Tyr Val Arg Phe Thr Pro Leu Leu Leu Val Leu 50 55 60 Ala Ala Leu Ala Ser Ala Gly Val Met Leu Trp Tyr Phe Leu Gly Tyr 65 70 75 80 Lys Ala Glu Val Thr Ile Ser Gin Val Tyr Ser Gly Ser Leu Arg Val 85 90 95 Leu Asn Arg His Phe Ser Gin Asp Leu Ala Arg Arg Glu Ser Ile Ala 100 105 110 Phe Arg Thr Glu Thr Ala Lys Ala Gin Lys Met Phe Gin Glu Leu Val 115 120 125 Ala Ser Thr Arg Leu Gly Thr Tyr Tyr Asn Ser Ser Ser Ile Tyr Ala 130 135 140 Phe Gly Glu Gly Pro Leu Ile Cys Phe Phe Trp Phe Ile Leu Asp Ile 145 150 155 160 Pro Glu Tyr Gin Arg Leu Thr Leu Ser Pro Glu Val Val Arg Glu Leu 165 170 175 Leu Val Gly Glu Leu Leu Ser Asn Ser Ser Ala Leu Ala Ser Tyr Arg 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 Arg Leu Arg 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 Leu Lys Val Gln 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 Leu His Ser Phe 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 Pro Gin 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 Gin Gin Tyr Asn Leu Leu Cys Thr Gin Gin 385 390 395 400 Gln Trp Met Ile Gin Asn Arg Arg Leu Cys Gly Phe Arg Thr Leu Gin 405 410 415 Pro Tyr Ala Glu Arg Ile Pro Val Val Ala Ser Asp Gly Ile Thr Ile 420 425 430 Asn Phe Thr Ser Gin Ile Ser Leu Thr Gly Pro Gly Val Gin Val Tyr 435 440 445 Tyr Ser Leu Tyr Asn Gin 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 Gin 485 490 495 Cys Gin Glu Asp Ser Thr Cys lie Ser Leu Pro Arg Val Cys Asp Arg 500 505 510 Gln Pro Asp Cys Leu Asn Gly Ser Asp Glu Glu Gin Cys Gin Glu Gly 515 520 525 Val Pro Cys Gly Thr Phe Thr Phe Gin Cys Glu Asp Arg Ser Cys Val 530 535 540 Lys Lys Pro Asn Pro Glu Cys Asp Gly Gin Ala Asp Cys Arg Asp Gly 545 550 555 560 Ser Asp Glu Glu His Cys Asp Cys Gly Leu Gin Gly Pro Ser Ser Arg 565 570 575 Ile Val Gly Gly Ala Met Ser Ser Glu Gly Glu Trp Pro Trp Gin Ala 580 585 590 Ser Leu Gin lie Arg Gly Arg His lie Cys Gly Gly Ala Leu lie Ala 595 600 605 Asp Arg Trp Val lie Thr Ala Ala His Cys Phe Gin Glu Asp Ser Met 610 615 620 Ala Ser Pro Arg Leu Trp Thr Val Phe Leu Gly Lys Met Arg Gin 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 Gly Ser Ser Thr Leu 705 710 715 720 Gln Lys Val Asp Val Gln Leu Ile Pro Gln Asp Leu Cys Asn Glu Ala 725 730 735 Tyr Arg Tyr Gln Val Thr 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 Lys 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 Val Asn Trp Ile Gln Gln Val Leu Thr 805 810 <210> 6 <211> 800 <212> PRT <213> Cynomolgus monkey <400> 6 Met Pro Val Ala Lys Ala Pro Gin Val Ala Gly Gly Gin 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 Gin Val Tyr Ser Gly Ser 65 70 75 80 Leu Arg Val Leu Asn Arg His Phe Ser Gin Asp Leu Thr Arg Arg Glu 85 90 95 Ser Ser Ala Phe Arg Ser Glu Thr Ala Lys Ala Gin 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 Gin lie 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 lie Ala Ala Leu Asn Ser Thr Leu 195 200 205 Gly Cys Tyr Arg Tyr Ser Tyr Val Gly Gin Gly Gin Val Leu Arg Leu 210 215 220 Lys Gly Pro Asp His Leu Ala Ser Ser Cys Leu Trp His Leu Gin 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 Gin 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 Gin Cys Gin Glu Asp Ser Thr Cys He Ser Leu Leu 485 490 495 Lys Val Cys Asp Gly Gin Pro Asp Cys Leu Asn Gly Ser Asp Glu Glu 500 505 510 Arg Cys Gin Glu Gly Val Pro Cys Gly Thr Phe Thr Phe Gin Cys Glu 515 520 525 Asp Gin Ser Cys Val Lys Lys Pro Asn Pro Gin Cys Asp Gly Arg Pro 530 535 540 Asp Cys Arg Asp Gly Ser Asp Glu Gin His Cys Asp Cys Gly Leu Gin 545 550 555 560 Gly Pro Ser Ser Arg He Val Gly Gly Ala Val Ser Ser Glu Gly Glu 565 570 575 Trp Pro Trp Gin Ala Ser Leu Gin Val Arg Gly Arg His He Cys Gly 580 585 590 Gly Ala Leu He Ala Asp Arg Trp Val He 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 Gin 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 Gin 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 Gin Lys Val Asp Val Gin Leu Ile Pro Gin Asp 705 710 715 720 Leu Cys Ser Glu Ala Tyr Arg Tyr Gin Val Thr Pro Arg Met Leu Cys 725 730 735 Ala Gly Tyr Arg Lys Gly Lys Lys Asp Ala Cys Gin 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 <210> 7 <211> 252 <212> PRT <213> Mus Musculus <400> 7 Met Ala Gln Leu Cys Glu Leu Arg Arg Gly Arg Ala Leu Leu Ala Leu 1 5 10 15 Val Ala Ser Leu Leu Leu Ser Gly Ala Gln Val Ala Ser Arg Glu Leu 20 25 30 Asp Val His Glu Ser Cys Gly Val Ser Lys Val Val Gly Lys Cys Arg 35 40 45 Ala Ser Ile Pro Arg Trp Trp Tyr Asn Ile Thr Asp Gly Ser Cys Gln 50 55 60 Pro Phe Val Tyr Gly Gly Cys Glu Gly Asn Gly Asn Asn Tyr Gln Ser 65 70 75 80 Lys Glu Glu Cys Leu Asp Lys Cys Ala Gly Val Thr Glu Asn Thr Thr 85 90 95 Asp Asp Met Ala Arg Asn Arg Asn Gly Ala Asp Ser Ser Val Leu Ser 100 105 110 Val Pro Arg Lys Gin Ser Ala Glu Asp Leu Ser Ala Glu lie Phe Asn 115 120 125 Tyr Glu Glu Tyr Cys Val Pro Lys Ala Val Thr Gly Pro Cys Arg Ala 130 135 140 Ala Phe Pro Arg Trp Tyr Tyr Asp Thr Glu Lys Asn Ser Cys lie Ser 145 150 155 160 Phe lie Tyr Gly Gly Cys Arg Gly Asn Lys Asn Ser Tyr Leu Ser Gin 165 170 175 Glu Ala Cys Met Gin His Cys Ser Gly Lys Gin Met His Pro Phe Leu 180 185 190 Thr Pro Gly Leu Lys Ala Val lie Leu Val Gly Leu Phe Leu Met Val 195 200 205 Leu lie Leu Leu Leu Gly Thr Ser Met Val Cys Leu lie Arg Val Val 210 215 220 Arg Arg Lys Gin Glu Arg Ala Leu Arg Thr Val Trp Ser Thr Ala Asp 225 230 235 240 Asp Lys Glu Gin Leu Val Lys Asn Thr Cys Val Leu 245 250 <210> 8 <211> 754 <212> PRT <213> Homo sapiens <400> 8 Tyr Lys Ala Glu Val Met Val Ser Gln Val Tyr Ser Gly Ser Leu Arg 1 5 10 15 Val Leu Asn Arg His Phe Ser Gln Asp Leu Thr Arg Arg Glu Ser Ser 20 25 30 Ala Phe Arg Ser Glu Thr Ala Lys Ala Gln Lys Met Leu Lys Glu Leu 35 40 45 Ile Thr Ser Thr Arg Leu Gly Thr Tyr Tyr Asn Ser Ser Ser Val Tyr 50 55 60 Ser Phe Gly Glu Gly Pro Leu Thr Cys Phe Phe Trp Phe Ile Leu Gln 65 70 75 80 Ile Pro Glu His Arg Arg Leu Met Leu Ser Pro Glu Val Val Gln Ala 85 90 95 Leu Leu Val Glu Glu Leu Leu Ser Thr Val Asn Ser Ser Ala Ala Val 100 105 110 Pro Tyr Arg Ala Glu Tyr Glu Val Asp Pro Glu Gly Leu Val Ile Leu 115 120 125 Glu Ala Ser Val Lys Asp Ile Ala Ala Leu Asn Ser Thr Leu Gly Cys 130 135 140 Tyr Arg Tyr Ser Tyr Val Gly Gln Gly Gln Val Leu Arg Leu Lys Gly 145 150 155 160 Pro Asp His Leu Ala Ser Ser Cys Leu Trp His Leu Gln Gly Pro Lys 165 170 175 Asp Leu Met Leu Lys Leu Arg Leu Glu Trp Thr Leu Ala Glu Cys Arg 180 185 190 Asp Arg Leu Ala Met Tyr Asp Val Ala Gly Pro Leu Glu Lys Arg Leu 195 200 205 Ile Thr Ser Val Tyr Gly Cys Ser Arg Gln Glu Pro Val Val Glu Val 210 215 220 Leu Ala Ser Gly Ala Ile Met Ala Val Val Trp Lys Lys Gly Leu His 225 230 235 240 Ser Tyr Tyr Asp Pro Phe Val Leu Ser Val Gln Pro Val Val Phe Gln 245 250 255 Ala Cys Glu Val Asn Leu Thr Leu Asp Asn Arg Leu Asp Ser Gln Gly 260 265 270 Val Leu Ser Thr Pro Tyr Phe Pro Ser Tyr Tyr Ser Pro Gln Thr His 275 280 285 Cys Ser Trp His Leu Thr Val Pro Ser Leu Asp Tyr Gly Leu Ala Leu 290 295 300 Trp Phe Asp Ala Tyr Ala Leu Arg Arg Gln Lys Tyr Asp Leu Pro Cys 305 310 315 320 Thr Gin Gly Gin Trp Thr He Gin Asn Arg Arg Leu Cys Gly Leu Arg 325 330 335 He Leu Gin Pro Tyr Ala Glu Arg He Pro Val Val Ala Thr Ala Gly 340 345 350 He Thr He Asn Phe Thr Ser Gin He Ser Leu Thr Gly Pro Gly Val 355 360 365 Arg Val His Tyr Gly Leu Tyr Asn Gin Ser Asp Pro Cys Pro Gly Glu 370 375 380 Phe Leu Cys Ser Val Asn Gly Leu Cys Val Pro Ala Cys Asp Gly Val 385 390 395 400 Lys Asp Cys Pro Asn Gly Leu Asp Glu Arg Asn Cys Val Cys Arg Ala 405 410 415 Thr Phe Gin Cys Lys Glu Asp Ser Thr Cys He Ser Leu Pro Lys Val 420 425 430 Cys Asp Gly Gin Pro Asp Cys Leu Asn Gly Ser Asp Glu Glu Gin Cys 435 440 445 Gln Glu Gly Val Pro Cys Gly Thr Phe Thr Phe Gin Cys Glu Asp Arg 450 455 460 Ser Cys Val Lys Lys Pro Asn Pro Gin Cys Asp Gly Arg Pro Asp Cys 465 470 475 480 Arg Asp Gly Ser Asp Glu Glu His Cys Asp Cys Gly Leu Gin Gly Pro 485 490 495 Ser Ser Arg He Val Gly Gly Ala Val Ser Ser Glu Gly Glu Trp Pro 500 505 510 Trp Gin Ala Ser Leu Gin Val Arg Gly Arg His He Cys Gly Gly Ala 515 520 525 Leu He Ala Asp Arg Trp Val He Thr Ala Ala His Cys Phe Gin Glu 530 535 540 Asp Ser Met Ala Ser Thr Val Leu Trp Thr Val Phe Leu Gly Lys Val 545 550 555 560 Trp Gin Asn Ser Arg Trp Pro Gly Glu Val Ser Phe Lys Val Ser Arg 565 570 575 Leu Leu Leu His Pro Tyr His Glu Glu Asp Ser His Asp Tyr Asp Val 580 585 590 Ala Leu Leu Gin Leu Asp His Pro Val Val Arg Ser Ala Ala Val Arg 595 600 605 Pro Val Cys Leu Pro Ala Arg Ser His Phe Phe Glu Pro Gly Leu His 610 615 620 Cys Trp lie Thr Gly Trp Gly Ala Leu Arg Glu Gly Gly Pro lie Ser 625 630 635 640 Asn Ala Leu Gin Lys Val Asp Val Gin Leu lie Pro Gin Asp Leu Cys 645 650 655 Ser Glu Val Tyr Arg Tyr Gin Val Thr Pro Arg Met Leu Cys Ala Gly 660 665 670 Tyr Arg Lys Gly Lys Lys Asp Ala Cys Gin Gly Asp Ser Gly Gly Pro 675 680 685 Leu Val Cys Lys Ala Leu Ser Gly Arg Trp Phe Leu Ala Gly Leu Val 690 695 700 Ser Trp Gly Leu Gly Cys Gly Arg Pro Asn Tyr Phe Gly Val Tyr Thr 705 710 715 720 Arg lie Thr Gly Val lie Ser Trp lie Gin Gin Val Val Thr Gly Lys 725 730 735 Pro lie Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr His His His His 740 745 750 His His <210> 9 <211> 1058 <212> PRT <213> Artificial Sequence <220> <223> Human Proteinase-2 Antigen eGFP / flag Tag <400> 9 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Val Asn Phe Lys lie Arg His Asn lie Glu Asp Gly Ser 165 170 175 Val Gln Leu Ala Asp His Tyr Gin Gin Asn Thr Pro lie Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gin Ser Ala Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Met 225 230 235 240 Leu Leu Leu Phe His Ser Lys Arg Met Pro Val Ala Glu Ala Pro Gin 245 250 255 Val Ala Gly Gly Gin Gly Asp Gly Gly Asp Gly Glu Glu Ala Glu Pro 260 265 270 Glu Gly Met Phe Lys Ala Cys Glu Asp Ser Lys Arg Lys Ala Arg Gly 275 280 285 Tyr Leu Arg Leu Val Pro Leu Phe Val Leu Leu Ala Leu Leu Val Leu 290 295 300 Ala Ser Ala Gly Val Leu Leu Trp Tyr Phe Leu Gly Tyr Lys Ala Glu 305 310 315 320 Val Met Val Ser Gin Val Tyr Ser Gly Ser Leu Arg Val Leu Asn Arg 325 330 335 His Phe Ser Gin Asp Leu Thr Arg Arg Glu Ser Ser Ala Phe Arg Ser 340 345 350 Glu Thr Ala Lys Ala Gin Lys Met Leu Lys Glu Leu He Thr Ser Thr 355 360 365 Arg Leu Gly Thr Tyr Tyr Asn Ser Ser Ser Val Tyr Ser Phe Gly Glu 370 375 380 Gly Pro Leu Thr Cys Phe Phe Trp Phe He Leu Gin He Pro Glu His 385 390 395 400 Arg Arg Leu Met Leu Ser Pro Glu Val Val Gin Ala Leu Leu Val Glu 405 410 415 Glu Leu Leu Ser Thr Val Asn Ser Ser Ala Ala Val Pro Tyr Arg Ala 420 425 430 Glu Tyr Glu Val Asp Pro Glu Gly Leu Val He Leu Glu Ala Ser Val 435 440 445 Lys Asp He Ala Ala Leu Asn Ser Thr Leu Gly Cys Tyr Arg Tyr Ser 450 455 460 Tyr Val Gly Gin Gly Gin Val Leu Arg Leu Lys Gly Pro Asp His Leu 465 470 475 480 Ala Ser Ser Cys Leu Trp His Leu Gin Gly Pro Lys Asp Leu Met Leu 485 490 495 Lys Leu Arg Leu Gin Trp Thr Leu Ala Gin Cys Arg Asp Arg Leu Ala 500 505 510 Met Tyr Asp Val Ala Gly Pro Leu Gin Lys Arg Leu He Thr Ser Val 515 520 525 Tyr Gly Cys Ser Arg Gin Gin Pro Val Val Gin Val Leu Ala Ser Gly 530 535 540 Ala He Met Ala Val Val Trp Lys Lys Gly Leu His Ser Tyr Tyr Asp 545 550 555 560 Pro Phe Val Leu Ser Val Gin Pro Val Val Phe Gin Ala Cys Gin Val 565 570 575 Asn Leu Thr Leu Asp Asn Arg Leu Asp Ser Gin Gly Val Leu Ser Thr 580 585 590 Pro Tyr Phe Pro Ser Tyr Tyr Ser Pro Gin Thr His Cys Ser Trp His 595 600 605 Leu Thr Val Pro Ser Leu Asp Tyr Gly Leu Ala Leu Trp Phe Asp Ala 610 615 620 Tyr Ala Leu Arg Arg Gin Lys Tyr Asp Leu Pro Cys Thr Gin Gly Gin 625 630 635 640 Trp Thr Ile Gin Asn Arg Arg Leu Cys Gly Leu Arg Ile Leu Gin Pro 645 650 655 Tyr Ala Glu Arg Ile Pro Val Val Ala Thr Ala Gly Ile Thr Ile Asn 660 665 670 Phe Thr Ser Gin Ile Ser Leu Thr Gly Pro Gly Val Arg Val His Tyr 675 680 685 Gly Leu Tyr Asn Gin Ser Asp Pro Cys Pro Gly Glu Phe Leu Cys Ser 690 695 700 Val Asn Gly Leu Cys Val Pro Ala Cys Asp Gly Val Lys Asp Cys Pro 705 710 715 720 Asn Gly Leu Asp Glu Arg Asn Cys Val Cys Arg Ala Thr Phe Gin Cys 725 730 735 Lys Gin Asp Ser Thr Cys Ile Ser Leu Pro Lys Val Cys Asp Gly Gin 740 745 750 Pro Asp Cys Leu Asn Gly Ser Asp Glu Glu Gin Cys Gin Glu Gly Val 755 760 765 Pro Cys Gly Thr Phe Thr Phe Gin Cys Glu Asp Arg Ser Cys Val Lys 770 775 780 Lys Pro Asn Pro Gin Cys Asp Gly Arg Pro Asp Cys Arg Asp Gly Ser 785 790 795 800 Asp Glu Glu His Cys Asp Cys Gly Leu Gin Gly Pro Ser Ser Arg Ile 805 810 815 Val Gly Gly Ala Val Ser Ser Glu Gly Glu Trp Pro Trp Gin Ala Ser 820 825 830 Leu Gin Val Arg Gly Arg His Ile Cys Gly Gly Ala Leu Ile Ala Asp 835 840 845 Arg Trp Val Ile Thr Ala Ala His Cys Phe Gin Glu Asp Ser Met Ala 850 855 860 Ser Thr Val Leu Trp Thr Val Phe Leu Gly Lys Val Trp Gin Asn Ser 865 870 875 880 Arg Trp Pro Gly Glu Val Ser Phe Lys Val Ser Arg Leu Leu Leu His 885 890 895 Pro Tyr His Glu Glu Asp Ser His Asp Tyr Asp Val Ala Leu Leu Gin 900 905 910 Leu Asp His Pro Val Val Arg Ser Ala Ala Val Arg Pro Val Cys Leu 915 920 925 Pro Ala Arg Ser His Phe Phe Glu Pro Gly Leu His Cys Trp Ile Thr 930 935 940 Gly Trp Gly Ala Leu Arg Glu Gly Gly Pro Ile Ser Asn Ala Leu Gin 945 950 955 960 Lys Val Asp Val Gin Leu lie Pro Gin Asp Leu Cys Ser Glu Val Tyr 965 970 975 Arg Tyr Gin Val Thr Pro Arg Met Leu Cys Ala Gly Tyr Arg Lys Gly 980 985 990 Lys Lys Asp Ala Cys Gin Gly Asp Ser Gly Gly Pro Leu Val Cys Lys 995 1000 1005 Ala Leu Ser Gly Arg Trp Phe Leu Ala Gly Leu Val Ser Trp Gly 1010 1015 1020 Leu Gly Cys Gly Arg Pro Asn Tyr Phe Gly Val Tyr Thr Arg lie 1025 1030 1035 Thr Gly Val lie Ser Trp lie Gin Gin Val Val Thr Asp Tyr Lys 1040 1045 1050 Asp Asp Asp Asp Lys 1055 <210> 10 <211> 802 <212> PRT <213> Homo sapiens <400> 10 Met Pro Val Ala Glu Ala Pro Gin Val Ala Gly Gly Gin 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 Gin Gly Val Leu Ser Thr Pro Tyr Phe Pro Ser Tyr Tyr Ser 340 345 350 Pro Gin 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 Gin Leu Tyr 370 375 380 Asp Leu Pro Cys Thr Gin Gly Gin Trp Thr He Gin Asn Arg Arg Leu 385 390 395 400 Cys Gly Leu Arg He Leu Gin Pro Tyr Ala Glu Arg He Pro Val Val 405 410 415 Ala Thr Ala Gly He Thr He Asn Phe Thr Ser Gin He Ser Leu Thr 420 425 430 Gly Pro Gly Val Arg Val His Tyr Gly Leu Tyr Asn Gin 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 Gin Cys Lys Glu Asp Ser Thr Cys He Ser 485 490 495 Leu Pro Lys Val Cys Asp Gly Gin Pro Asp Cys Leu Asn Gly Ser Asp 500 505 510 Glu Glu Gin Cys Gin Glu Gly Val Pro Cys Gly Thr Phe Thr Phe Gin 515 520 525 Cys Glu Asp Arg Ser Cys Val Lys Lys Pro Asn Pro Gin 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 Gin Gly Pro Ser Ser Arg He Val Gly Gly Ala Val Ser Ser Glu 565 570 575 Gly Glu Trp Pro Trp Gin Ala Ser Leu Gin Val Arg Gly Arg His He 580 585 590 Cys Gly Gly Ala Leu He Ala Asp Arg Trp Val He Thr Ala Ala His 595 600 605 Cys Phe Gin Glu Asp Ser Met Ala Ser Thr Val Leu Trp Thr Val Phe 610 615 620 Leu Gly Lys Val Trp Gin 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 <210> 11 <211> 802 <212> PRT <213> Homo sapiens <400> 11 Met Pro Val Ala Glu Ala Pro Gin Val Ala Gly Gly Gin 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 Gin Val Tyr Ser 65 70 75 80 Gly Ser Leu Arg Val Leu Asn Arg His Phe Ser Gin Asp Leu Thr Arg 85 90 95 Arg Glu Ser Ser Ala Phe Arg Ser Glu Thr Ala Lys Ala Gin 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 lie Leu Gin lie Pro Glu His Arg Arg Leu Met Leu Ser Pro Glu 145 150 155 160 Val Val Gin 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 lie Leu Glu Ala Ser Val Lys Asp lie Ala Ala Leu Asn Ser 195 200 205 Thr Leu Gly Cys Tyr Arg Tyr Ser Tyr Val Gly Gin Gly Gin 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 Glu 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 lie Thr Ser Val Tyr Gly Cys Ser Arg Gin Glu Pro 275 280 285 Val Val Glu Val Leu Ala Ser Gly Ala lie 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 Gin 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 Gin 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 Gin Lys Val Asp Val Gin Leu Ile Pro 705 710 715 720 Gln Asp Leu Cys Ser Glu Val Tyr Arg Tyr Gin Val Thr Pro Arg Met 725 730 735 Leu Cys Ala Gly Tyr Arg Lys Gly Lys Lys Asp Ala Cys Gin 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 <210> 12 <211> 802 <212> PRT <213> Homo sapiens <400> 12 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 lie Thr lie Asn Phe Thr Ser Gin lie Ser Leu Thr 420 425 430 Gly Pro Gly Val Arg Val His Tyr Gly Leu Tyr Asn Gin 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 Gin Cys Lys Glu Asp Ser Thr Cys lie Ser 485 490 495 Leu Pro Lys Val Cys Asp Gly Gin Pro Asp Cys Leu Asn Gly Ser Asp 500 505 510 Glu Glu Gin Cys Gin Glu Gly Val Pro Cys Gly Thr Phe Thr Phe Gin 515 520 525 Cys Glu Asp Arg Ser Cys Val Lys Lys Pro Asn Pro Gin 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 Gin Gly Pro Ser Ser Arg lie 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 Ala 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 <210> 13 <211> 802 <212> PRT <213> Homo sapiens <400> 13 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 Gin Val Tyr Ser 65 70 75 80 Gly Ser Leu Arg Val Leu Asn Arg His Phe Ser Gin Asp Leu Thr Arg 85 90 95 Arg Glu Ser Ser Ala Phe Arg Ser Glu Thr Ala Lys Ala Gin Lys Met 100 105 110 Leu Lys Glu Leu lie 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 lie Leu Gin lie Pro Glu His Arg Arg Leu Met Leu Ser Pro Glu 145 150 155 160 Val Val Gin 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 lie Leu Glu Ala Ser Val Lys Asp lie Ala Ala Leu Asn Ser 195 200 205 Thr Leu Gly Cys Tyr Arg Tyr Ser Tyr Val Gly Gin Gly Gin 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 Glu 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 Gin Gly Gin Trp Thr He Gin Asn Arg Arg Leu 385 390 395 400 Cys Gly Leu Arg He Leu Gin Pro Tyr Ala Glu Arg He Pro Val Val 405 410 415 Ala Thr Ala Gly He Thr He Asn Phe Thr Ser Gin He Ser Leu Thr 420 425 430 Gly Pro Gly Val Arg Val His Tyr Gly Leu Tyr Asn Gin 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 Gin Cys Lys Glu Asp Ser Thr Cys He Ser 485 490 495 Leu Pro Lys Val Cys Asp Gly Gin Pro Asp Cys Leu Asn Gly Ser Asp 500 505 510 Glu Glu Gin Cys Gin Glu Gly Val Pro Cys Gly Thr Phe Thr Phe Gin 515 520 525 Cys Glu Asp Arg Ser Cys Val Lys Lys Pro Asn Pro Gin 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 Gin Lys Val Asp Val Gin Leu Ile Pro 705 710 715 720 Gln Asp Leu Cys Ser Glu Ala Tyr Arg Tyr Gin Val Thr Pro Arg Met 725 730 735 Leu Cys Ala Gly Tyr Arg Lys Gly Lys Lys Asp Ala Cys Gin 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 Gin Gin Val 785 790 795 800 Val Thr <210> 14 <211> 866 <212> PRT <213> Artificial Sequence <220> <223> Human Proteinase K-1 Antigen His Tag <400> 14 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 Gin Gly Cys Ser Cys Pro Ala Gin 515 520 525 Thr Phe Arg Cys Ser Asn Gly Lys Cys Leu Ser Lys Ser Gin Gin 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 Gin Ala Arg Val Val Gly Gly Thr Asp Ala Asp Glu Gly 610 615 620 Glu Trp Pro Trp Gin Val Ser Leu His Ala Leu Gly Gin Gly His Ile 625 630 635 640 Cys Gly Ala Ser Leu Ile Ser Pro Asn Trp Leu Val Ser Ala Ala His 645 650 655 Cys Tyr lie Asp Asp Arg Gly Phe Arg Tyr Ser Asp Pro Thr Gin Trp 660 665 670 Thr Ala Phe Leu Gly Leu His Asp Gin Ser Gin Arg Ser Ala Pro Gly 675 680 685 Val Gin Glu Arg Arg Leu Lys Arg lie lie Ser His Pro Phe Phe Asn 690 695 700 Asp Phe Thr Phe Asp Tyr Asp lie Ala Leu Leu Glu Leu Glu Lys Pro 705 710 715 720 Ala Glu Tyr Ser Ser Met Val Arg Pro lie Cys Leu Pro Asp Ala Ser 725 730 735 His Val Phe Pro Ala Gly Lys Ala lie Trp Val Thr Gly Trp Gly His 740 745 750 Thr Gin Tyr Gly Gly Thr Gly Ala Leu lie Leu Gin Lys Gly Glu lie 755 760 765 Arg Val lie Asn Gin Thr Thr Cys Glu Asn Leu Leu Pro Gin Gin lie 770 775 780 Thr Pro Arg Met Met Cys Val Gly Phe Leu Ser Gly Gly Val Asp Ser 785 790 795 800 Cys Gin Gly Asp Ser Gly Gly Pro Leu Ser Ser Val Glu Ala Asp Gly 805 810 815 Arg Ile Phe Gin Ala Gly Val Val Ser Trp Gly Asp Gly Cys Ala Gin 820 825 830 Arg Asn Lys Pro Gly Val Tyr Thr Arg Leu Pro Leu Phe Arg Asp Trp 835 840 845 Ile Lys Glu Asn Thr Gly Val Gly Gly Gly Gly Ser His His His His 850 855 860 His His 865 <210> 15 <211> 854 <212> PRT <213> Artificial Sequence <220> <223> Human Proteinase-3 Antigen His Tag <400> 15 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 Gin Val Val Ser Ala Gin 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 Gin Arg Pro Ile Gly Lys Ala Lys Pro Lys Lys Gin Ser 50 55 60 Lys Lys Lys Val Pro Phe Trp Asn Val Gin Asn Lys 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 Gin Asn Cys Thr Gin Ser lie Pro Cys Asn 545 550 555 560 Asn Arg Thr Phe Lys Cys Gly Asn Asp lie Cys Phe Arg Lys Gin 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 lie lie Gly 595 600 605 Gly Thr Asp Thr Leu Glu Gly Gly Trp Pro Trp Gin Val Ser Leu His 610 615 620 Phe Val Gly Ser Ala Tyr Cys Gly Ala Ser Val lie 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 Gin Gly Asn Ala Lys 660 665 670 Phe Val Ser Pro Val Arg Arg lie Val Val His Glu Tyr Tyr Asn Ser 675 680 685 Gln Thr Phe Asp Tyr Asp lie Ala Leu Leu Gin Leu Ser lie 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 Gly Gly Gly Gly Ser 835 840 845 His His His His His His 850 <210> 16 <211> 1058 <212> PRT <213> Artificial Sequence <220> <223> Mouse proteinase-2 antigen eGFP / flag tag <400> 16 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Met 225 230 235 240 Pro Arg Cys Phe Gln Leu Pro Cys Ser Thr Arg Met Pro Thr Thr Glu 245 250 255 Val Pro Gln Ala Ala Asp Gly Gln Gly Asp Ala Gly Asp Gly Glu Glu 260 265 270 Ala Ala Glu Pro Glu Gly Lys Phe Lys Pro Pro Lys Asn Thr Lys Arg 275 280 285 Lys Asn Arg Asp Tyr Val Arg Phe Thr Pro Leu Leu Leu Val Leu Ala 290 295 300 Ala Leu Val Ser Ala Gly Val Met Leu Trp Tyr Phe Leu Gly Tyr Lys 305 310 315 320 Ala Glu Val Thr Val Ser Gln Val Tyr Ser Gly Ser Leu Arg Val Leu 325 330 335 Asn Arg His Phe Ser Gln Asp Leu Gly Arg Arg Glu Ser Ile Ala Phe 340 345 350 Arg Ser Glu Ser Ala Lys Ala Gln Lys Met Leu Gln Glu Leu Val Ala 355 360 365 Ser Thr Arg Leu Gly Thr Tyr Tyr Asn Ser Ser Ser Val Tyr Ser Phe 370 375 380 Gly Glu Gly Pro Leu Thr Cys Phe Phe Trp Phe Ile Leu Asp Ile Pro 385 390 395 400 Glu Tyr Gln Arg Leu Thr Leu Ser Pro Glu Val Val Arg Glu Leu Leu 405 410 415 Val Asp Glu Leu Leu Ser Asn Ser Ser Thr Leu Ala Ser Tyr Lys Thr 420 425 430 Glu Tyr Glu Val Asp Pro Glu Gly Leu Val Ile Leu Glu Ala Ser Val 435 440 445​​​​​​​​​​​​​​​​​​​ Asn Asp Ile Val Val Leu Asn Ser Thr Leu Gly Cys Tyr Arg Tyr Ser 450 455 460 Tyr Val Asn Pro Gly Gln Val Leu Pro Leu Lys Gly Pro Asp Gln Gln 465 470 475 480 Thr Thr Ser Cys Leu Trp His Leu Gln Gly Pro Glu Asp Leu Met Ile 485 490 495 Lys Val Arg Leu Glu Trp Thr Arg Val Asp Cys Arg Asp Arg Val Ala 500 505 510 Met Tyr Asp Ala Ala Gly Pro Leu Glu Lys Arg Leu Ile Thr Ser Val 515 520 525 Tyr Gly Cys Ser Arg Gln Glu Pro Val Met Glu Val Leu Ala Ser Gly 530 535 540 Ser Val Met Ala Val Val Trp Lys Lys Gly Met His Ser Tyr Tyr Asp 545 550 555 560 Pro Phe Leu Leu Ser Val Lys Ser Val Ala Phe Gln Asp Cys Gln Val 565 570 575 Asn Leu Thr Leu Glu Gly Arg Leu Asp Thr Gln Gly Phe Leu Arg Thr 580 585 590 Pro Tyr Tyr Pro Ser Tyr Tyr Ser Pro Ser Thr His Cys Ser Trp His 595 600 605 Leu Thr Val Pro Ser Leu Asp Tyr Gly Leu Ala Leu Trp Phe Asp Ala 610 615 620 Tyr Ala Leu Arg Arg Gln Lys Tyr Asn Arg Leu Cys Thr Gln Gly Gln 625 630 635 640 Trp Met Ile Gln Asn Arg Arg Leu Cys Gly Phe Arg Thr Leu Gln Pro 645 650 655 Tyr Ala Glu Arg Ile Pro Met Val Ala Ser Asp Gly Val Thr Ile Asn 660 665 670 Phe Thr Ser Gln Ile Ser Leu Thr Gly Pro Gly Val Gln Val Tyr Tyr 675 680 685 Ser Leu Tyr Asn Gln Ser Asp Pro Cys Pro Gly Glu Phe Leu Cys Ser 690 695 700 Val Asn Gly Leu Cys Val Pro Ala Cys Asp Gly Ile Lys Asp Cys Pro 705 710 715 720 Asn Gly Leu Asp Glu Arg Asn Cys Val Cys Arg Ala Met Phe Gln Cys 725 730 735 Gln Glu Asp Ser Thr Cys Ile Ser Leu Pro Arg Val Cys Asp Arg Gln 740 745 750 Pro Asp Cys Leu Asn Gly Ser Asp Glu Glu Gln Cys Gln Glu Gly Val 755 760 765 Pro Cys Gly Thr Phe Thr Phe Gin Cys Glu Asp Arg Ser Cys Val Lys 770 775 780 Lys Pro Asn Pro Gin Cys Asp Gly Gin Ser Asp Cys Arg Asp Gly Ser 785 790 795 800 Asp Glu Gin His Cys Asp Cys Gly Leu Gin Gly Leu Ser Ser Arg He 805 810 815 Val Gly Gly Thr Val Ser Ser Glu Gly Glu Trp Pro Trp Gin Ala Ser 820 825 830 Leu Gin He Arg Gly Arg His He Cys Gly Gly Ala Leu He Ala Asp 835 840 845 Arg Trp Val He Thr Ala Ala His Cys Phe Gin Glu Asp Ser Met Ala 850 855 860 Ser Pro Lys Leu Trp Thr Val Phe Leu Gly Lys Met Arg Gin Asn Ser 865 870 875 880 Arg Trp Pro Gly Glu Val Ser Phe Lys Val Ser Arg Leu Phe Leu His 885 890 895 Pro Tyr His Glu Glu Asp Ser His Asp Tyr Asp Val Ala Leu Leu Gin 900 905 910 Leu Asp His Pro Val Val Tyr Ser Ala Thr Val Arg Pro Val Cys Leu 915 920 925 Pro Ala Arg Ser His Phe Phe Glu Pro Gly Gln His Cys Trp Ile Thr 930 935 940 Gly Trp Gly Ala Gln Arg Glu Gly Gly Pro Val Ser Asn Thr Leu Gln 945 950 955 960 Lys Val Asp Val Gln Leu Val Pro Gln Asp Leu Cys Ser Glu Ala Tyr 965 970 975 Arg Tyr Gln Val Ser Pro Arg Met Leu Cys Ala Gly Tyr Arg Lys Gly 980 985 990 Lys Lys Asp Ala Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Arg 995 1000 1005 Glu Pro Ser Gly Arg Trp Phe Leu Ala Gly Leu Val Ser Trp Gly 1010 1015 1020 Leu Gly Cys Gly Arg Pro Asn Phe Phe Gly Val Tyr Thr Arg Val 1025 1030 1035 Thr Arg Val Ile Asn Trp Ile Gln Gln Val Leu Thr Asp Tyr Lys 1040 1045 1050 Asp Asp Asp Asp Lys 1055 <210> 17 <211> 799 <212> PRT <213> Mus Musculus <400> 17 Met Pro Thr Thr Glu Val Pro Gin Ala Ala Asp Gly Gin Gly Asp Ala 1 5 10 15 Gly Asp Gly Glu Glu Ala Ala Glu Pro Glu Gly Lys Phe Lys Pro Pro 20 25 30 Lys Asn Thr Lys Arg Lys Asn Arg Asp Tyr Val Arg Phe Thr Pro Leu 35 40 45 Leu Leu Val Leu Ala Ala Leu Val Ser Ala Gly Val Met Leu Trp Tyr 50 55 60 Phe Leu Gly Tyr Lys Ala Glu Val Thr Val Ser Gin Val Tyr Ser Gly 65 70 75 80 Ser Leu Arg Val Leu Asn Arg His Phe Ser Gin Asp Leu Gly Arg Arg 85 90 95 Glu Ser Ile Ala Phe Arg Ser Glu Ser Ala Lys Ala Gin Lys Met Leu 100 105 110 Gln Glu Leu Val Ala Ser Thr Arg Leu Gly Thr Tyr Tyr Asn Ser Ser 115 120 125 Ser Val Tyr Ser Phe Gly Glu Gly Pro Leu Thr Cys Phe Phe Trp Phe 130 135 140 Ile Leu Asp Ile Pro Glu Tyr Gin Arg Leu Thr Leu Ser Pro Glu Val 145 150 155 160 Val Arg Glu Leu Leu Val Asp Glu Leu Leu Ser Asn Ser Ser Thr Leu 165 170 175 Ala Ser Tyr Lys Thr Glu Tyr Glu Val Asp Pro Glu Gly Leu Val Ile 180 185 190 Leu Glu Ala Ser Val Asn Asp Ile Val Val Leu Asn Ser Thr Leu Gly 195 200 205 Cys Tyr Arg Tyr Ser Tyr Val Asn Pro Gly Gln Val Leu Pro Leu Lys 210 215 220 Gly Pro Asp Gln Gin Thr Thr Ser Cys Leu Trp His Leu Gin Gly Pro 225 230 235 240 Glu Asp Leu Met Ile Lys Val Arg Leu Glu Trp Thr Arg Val Asp Cys 245 250 255 Arg Asp Arg Val Ala Met Tyr Asp Ala Ala Gly Pro Leu Glu Lys Arg 260 265 270 Leu Ile Thr Ser Val Tyr Gly Cys Ser Arg Gin Glu Pro Val Met Glu 275 280 285 Val Leu Ala Ser Gly Ser Val Met Ala Val Val Trp Lys Lys Gly Met 290 295 300 His Ser Tyr Tyr Asp Pro Phe Leu Leu Ser Val Lys Ser Val Ala Phe 305 310 315 320 Gln Asp Cys Gln Val Asn Leu Thr Leu Glu Gly Arg Leu Asp Thr Gln 325 330 335 Gly Phe Leu Arg Thr Pro Tyr Tyr Pro Ser Tyr Tyr Ser Pro Ser Thr 340 345 350 His Cys Ser Trp His Leu Thr Val Pro Ser Leu Asp Tyr Gly Leu Ala 355 360 365 Leu Trp Phe Asp Ala Tyr Ala Leu Arg Arg Gln Lys Tyr Asn Arg Leu 370 375 380 Cys Thr Gln Gly Gln Trp Met Ile Gln Asn Arg Arg Leu Cys Gly Phe 385 390 395 400 Arg Thr Leu Gln Pro Tyr Ala Glu Arg Ile Pro Met Val Ala Ser Asp 405 410 415 Gly Val Thr Ile Asn Phe Thr Ser Gln Ile Ser Leu Thr Gly Pro Gly 420 425 430 Val Gln Val Tyr Tyr Ser Leu Tyr Asn Gln Ser Asp Pro Cys Pro Gly 435 440 445 Glu Phe Leu Cys Ser Val Asn Gly Leu Cys Val Pro Ala Cys Asp Gly 450 455 460 Ile Lys Asp Cys Pro Asn Gly Leu Asp Glu Arg Asn Cys Val Cys Arg 465 470 475 480 Ala Met Phe Gln Cys Gln Glu Asp Ser Thr Cys Ile Ser Leu Pro Arg 485 490 495 Val Cys Asp Arg Gln Pro Asp Cys Leu Asn Gly Ser Asp Glu Glu Gln 500 505 510 Cys Gln Glu Gly Val Pro Cys Gly Thr Phe Thr Phe Gln Cys Glu Asp 515 520 525 Arg Ser Cys Val Lys Lys Pro Asn Pro Glu Cys Asp Gly Gln Ser Asp 530 535 540 Cys Arg Asp Gly Ser Asp Glu Gln His Cys Asp Cys Gly Leu Gln Gly 545 550 555 560 Leu Ser Ser Arg Ile Val Gly Gly Thr Val Ser Ser Glu Gly Glu Trp 565 570 575 Pro Trp Gln Ala Ser Leu Gln Ile Arg Gly Arg His Ile Cys Gly Gly 580 585 590 Ala Leu Ile Ala Asp Arg Trp Val Ile Thr Ala Ala His Cys Phe Gln 595 600 605 Glu Asp Ser Met Ala Ser Pro Lys Leu Trp Thr Val Phe Leu Gly Lys 610 615 620 Met Arg Gin Asn Ser Arg Trp Pro Gly Glu Val Ser Phe Lys Val Ser 625 630 635 640 Arg Leu Phe Leu His Pro Tyr His Glu Glu Asp Ser His Asp Tyr Asp 645 650 655 Val Ala Leu Leu Gin Leu Asp His Pro Val Val Tyr Ser Ala Thr Val 660 665 670 Arg Pro Val Cys Leu Pro Ala Arg Ser His Phe Phe Glu Pro Gly Gin 675 680 685 His Cys Trp Ile Thr Gly Trp Gly Ala Gin Arg Glu Gly Gly Pro Val 690 695 700 Ser Asn Thr Leu Gin Lys Val Asp Val Gin Leu Val Pro Gin Asp Leu 705 710 715 720 Cys Ser Glu Ala Tyr Arg Tyr Gin Val Ser Pro Arg Met Leu Cys Ala 725 730 735 Gly Tyr Arg Lys Gly Lys Lys Asp Ala Cys Gin Gly Asp Ser Gly Gly 740 745 750 Pro Leu Val Cys Arg Glu Pro Ser Gly Arg Trp Phe Leu Ala Gly Leu 755 760 765 Val Ser Trp Gly Leu Gly Cys Gly Arg Pro Asn Phe Phe Gly Val Tyr 770 775 780 Thr Arg Val Thr Arg Val Ile Asn Trp Ile Gln Gln Val Leu Thr 785 790 795 <210> 18 <211> 799 <212> PRT <213> Mus norvegicus (Brown rat) <400> 18 Met Pro Thr Ala Glu Val Pro Gln Ala Ala Gly Gly Gln Gly Asp Gly 1 5 10 15 Gly Asp Gly Glu Glu Ala Ala Glu Pro Glu Gly Val Phe Lys Ala Pro 20 25 30 Arg Asn Ala Lys Arg Lys Asp Arg Asp Tyr Val Arg Phe Thr Pro Leu 35 40 45 Leu Leu Val Leu Ala Ala Leu Ala Ser Ala Gly Val Met Leu Trp Tyr 50 55 60 Phe Leu Gly Tyr Lys Ala Glu Val Thr Ile Ser Gln Val Tyr Ser Gly 65 70 75 80 Ser Leu Arg Val Leu Asn Arg His Phe Ser Gln Asp Leu Ala Arg Arg 85 90 95 Glu Ser Ile Ala Phe Arg Thr Glu Thr Ala Lys Ala Gln Lys Met Phe 100 105 110 Gln Glu Leu Val Ala Ser Thr Arg Leu Gly Thr Tyr Tyr Asn Ser Ser 115 120 125 Ser Ile Tyr Ala Phe Gly Glu Gly Pro Leu Ile Cys Phe Phe Trp Phe 130 135 140 Ile Leu Asp Ile Pro Glu Tyr Gln Arg Leu Thr Leu Ser Pro Glu Val 145 150 155 160 Val Arg Glu Leu Leu Val Gly Glu Leu Leu Ser Asn Ser Ser Ala Leu 165 170 175 Ala Ser Tyr Arg Thr Glu Tyr Glu Val Asp Pro Glu Gly Leu Val Ile 180 185 190 Leu Glu Ala Ser Val Asn Asp Ile Val Val Leu Asn Ser Thr Leu Gly 195 200 205 Cys Tyr Arg Tyr Ser Tyr Val Asn Pro Gly Gln Val Leu Arg Leu Arg 210 215 220 Gly Pro Asp Gln Gln Thr Thr Ser Cys Leu Trp His Leu Gln Gly Pro 225 230 235 240 Glu Asp Leu Met Leu Lys Val Gln Leu Glu Trp Thr Arg Val Asp Cys 245 250 255 Arg Asp Arg Val Ala Met Tyr Asp Ala Ala Gly Pro Leu Glu Lys Arg 260 265 270 Leu Ile Thr Ser Val Tyr Gly Cys Ser Arg Gin Glu Pro Val Met Glu 275 280 285 Val Leu Ala Ser Gly Ser Val Met Ala Val Val Trp Lys Lys Gly Leu 290 295 300 His Ser Phe Tyr Asp Pro Phe Leu Leu Ser Val Lys Ser Val Ala Phe 305 310 315 320 Gln Asp Cys Gin Val Asn Leu Thr Leu Glu Gly Arg Leu Asp Pro Gin 325 330 335 Gly Phe Leu Arg Thr Pro Tyr Tyr Pro Ser Tyr Tyr Ser Pro Ser Thr 340 345 350 His Cys Ser Trp His Leu Thr Val Pro Ser Leu Asp Tyr Gly Leu Ala 355 360 365 Leu Trp Phe Asp Ala Tyr Ala Leu Arg Arg Gin Lys Tyr Asn Leu Leu 370 375 380 Cys Thr Gin Gly Gin Trp Met Ile Gin Asn Arg Arg Leu Cys Gly Phe 385 390 395 400 Arg Thr Leu Gin Pro Tyr Ala Glu Arg Ile Pro Val Val Ala Ser Asp 405 410 415 Gly Ile Thr Ile Asn Phe Thr Ser Gin Ile Ser Leu Thr Gly Pro Gly 420 425 430 Val Gin Val Tyr Tyr Ser Leu Tyr Asn Gin Ser Asp Pro Cys Pro Gly 435 440 445 Glu Phe Leu Cys Ser Val Asn Gly Leu Cys Val Pro Ala Cys Asp Gly 450 455 460 Ile Lys Asp Cys Pro Asn Gly Leu Asp Glu Arg Asn Cys Val Cys Arg 465 470 475 480 Ala Met Phe Gin Cys Gin Glu Asp Ser Thr Cys Ile Ser Leu Pro Arg 485 490 495 Val Cys Asp Arg Gin Pro Asp Cys Leu Asn Gly Ser Asp Glu Glu Gin 500 505 510 Cys Gin Glu Gly Val Pro Cys Gly Thr Phe Thr Phe Gin Cys Glu Asp 515 520 525 Arg Ser Cys Val Lys Lys Pro Asn Pro Glu Cys Asp Gly Gin Ala Asp 530 535 540 Cys Arg Asp Gly Ser Asp Glu Glu His Cys Asp Cys Gly Leu Gin Gly 545 550 555 560 Pro Ser Ser Arg Ile Val Gly Gly Ala Met Ser Ser Glu Gly Glu Trp 565 570 575 Pro Trp Gin Ala Ser Leu Gin He Arg Gly Arg His He Cys Gly Gly 580 585 590 Ala Leu He Ala Asp Arg Trp Val He Thr Ala Ala His Cys Phe Gin 595 600 605 Glu Asp Ser Met Ala Ser Pro Arg Leu Trp Thr Val Phe Leu Gly Lys 610 615 620 Met Arg Gin Asn Ser Arg Trp Pro Gly Glu Val Ser Phe Lys Val Ser 625 630 635 640 Arg Leu Phe Leu His Pro Tyr His Glu Glu Asp Ser His Asp Tyr Asp 645 650 655 Val Ala Leu Leu Gin Leu Asp His Pro Val Val Tyr Ser Ala Thr Val 660 665 670 Arg Pro Val Cys Leu Pro Ala Arg Ser His Phe Phe Glu Pro Gly Gin 675 680 685 His Cys Trp He Thr Gly Trp Gly Ala Gin Arg Glu Gly Gly Pro Gly 690 695 700 Ser Ser Thr Leu Gin Lys Val Asp Val Gin Leu He Pro Gin Asp Leu 705 710 715 720 Cys Asn Glu Ala Tyr Arg Tyr Gin Val Thr Pro Arg Met Leu Cys Ala 725 730 735 Gly Tyr Arg Lys Gly Lys Lys Asp Ala Cys Gin Gly Asp Ser Gly Gly 740 745 750 Pro Leu Val Cys Lys Glu Pro Ser Gly Arg Trp Phe Leu Ala Gly Leu 755 760 765 Val Ser Trp Gly Leu Gly Cys Gly Arg Pro Asn Phe Phe Gly Val Tyr 770 775 780 Thr Arg Val Thr Arg Val Val Asn Trp Ile Gin Gin Val Leu Thr 785 790 795 <210> 19 <211> 800 <212> PRT <213> Macaca Nemaestrina <400> 19 Met Pro Val Ala Lys Ala Pro Gin Val Ala Gly Gly Gin 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 Gin Val Tyr Ser Gly Ser 65 70 75 80 Leu Arg Val Leu Asn Arg His Phe Ser Gin Asp Leu Thr Arg Arg Glu 85 90 95 Ser Ser Ala Phe Arg Ser Glu Thr Ala Lys Ala Gin Lys Met Leu Lys 100 105 110 Glu Leu He 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 He 130 135 140 Leu Gin He 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 He Leu Glu Ala Ser Val Lys Asp He Ala Ala Leu Asn Ser Thr Leu 195 200 205 Gly Cys Tyr Arg Tyr Ser Tyr Val Gly Gin Gly Gin Val Leu Arg Leu 210 215 220 Lys Gly Pro Asp His Leu Ala Ser Ser Cys Leu Trp His Leu Gin 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 Gin Pro Tyr Ala Glu Arg Ile Pro Val Val Ala Thr 405 410 415 Ala Gly Ile Thr Ile Asn Phe Thr Ser Gin Ile Ser Leu Thr Gly Pro 420 425 430 Gly Val Arg Val His Tyr Gly Leu Tyr Asn Gin 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 Gin Cys Gin Glu Asp Ser Thr Cys Ile Ser Leu Leu 485 490 495 Lys Val Cys Asp Gly Gin Pro Asp Cys Leu Asn Gly Ser Asp Glu Glu 500 505 510 Arg Cys Gin Glu Gly Val Pro Cys Gly Thr Phe Thr Phe Gin Cys Glu 515 520 525 Asp Gin Ser Cys Val Lys Lys Pro Asn Pro Gin Cys Asp Gly Arg Pro 530 535 540 Asp Cys Arg Asp Gly Ser Asp Glu Gin His Cys Asp Cys Gly Leu Gin 545 550 555 560 Gly Pro Ser Ser Arg lie Val Gly Gly Ala Val Ser Ser Glu Gly Glu 565 570 575 Trp Pro Trp Gin Ala Ser Leu Gin Val Arg Gly Arg His lie Cys Gly 580 585 590 Gly Ala Leu lie Ala Asp Arg Trp Val lie 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 Gin 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 Gin 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 lie Thr Gly Trp Gly Ala Leu Arg Glu Gly Gly Pro 690 695 700 Thr Ser Asn Ala Leu Gin Lys Val Asp Val Gin Leu He Pro Gin Asp 705 710 715 720 Leu Cys Ser Glu Ala Tyr Arg Tyr Gin Val Thr Pro Arg Met Leu Cys 725 730 735 Ala Gly Tyr Arg Lys Gly Lys Lys Asp Ala Cys Gin 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 He Thr Gly Val He Gly Trp He Gin Gin Val Val Thr 785 790 795 800 <210> 20 <211> 756 <212> PRT <213> Artificial Sequence <220> <223> Human Proteinase-2 ECD Protein His Tag <400> 20 Tyr Lys Ala Glu Val Met Val Ser Gin Val Tyr Ser Gly Ser Leu Arg 1 5 10 15 Val Leu Asn Arg His Phe Ser Gin Asp Leu Thr Arg Arg Glu Ser Ser 20 25 30 Ala Phe Arg Ser Glu Thr Ala Lys Ala Gln Lys Met Leu Lys Glu Leu 35 40 45 Ile Thr Ser Thr Arg Leu Gly Thr Tyr Tyr Asn Ser Ser Ser Val Tyr 50 55 60 Ser Phe Gly Glu Gly Pro Leu Thr Cys Phe Phe Trp Phe Ile Leu Gln 65 70 75 80 Ile Pro Glu His Arg Arg Leu Met Leu Ser Pro Glu Val Val Gln Ala 85 90 95 Leu Leu Val Glu Glu Leu Leu Ser Thr Val Asn Ser Ser Ala Ala Val 100 105 110 Pro Tyr Arg Ala Glu Tyr Glu Val Asp Pro Glu Gly Leu Val Ile Leu 115 120 125 Glu Ala Ser Val Lys Asp Ile Ala Ala Leu Asn Ser Thr Leu Gly Cys 130 135 140 Tyr Arg Tyr Ser Tyr Val Gly Gln Gly Gln Val Leu Arg Leu Lys Gly 145 150 155 160 Pro Asp His Leu Ala Ser Ser Cys Leu Trp His Leu Gln Gly Pro Lys 165 170 175 Asp Leu Met Leu Lys Leu Arg Leu Glu Trp Thr Leu Ala Glu Cys Arg 180 185 190 Asp Arg Leu Ala Met Tyr Asp Val Ala Gly Pro Leu Glu Lys Arg Leu 195 200 205 Ile Thr Ser Val Tyr Gly Cys Ser Arg Gln Glu Pro Val Val Glu Val 210 215 220 Leu Ala Ser Gly Ala Ile Met Ala Val Val Trp Lys Lys Gly Leu His 225 230 235 240 Ser Tyr Tyr Asp Pro Phe Val Leu Ser Val Gln Pro Val Val Phe Gln 245 250 255 Ala Cys Glu Val Asn Leu Thr Leu Asp Asn Arg Leu Asp Ser Gln Gly 260 265 270 Val Leu Ser Thr Pro Tyr Phe Pro Ser Tyr Tyr Ser Pro Gln Thr His 275 280 285 Cys Ser Trp His Leu Thr Val Pro Ser Leu Asp Tyr Gly Leu Ala Leu 290 295 300 Trp Phe Asp Ala Tyr Ala Leu Arg Arg Gln Lys Tyr Asp Leu Pro Cys 305 310 315 320 Thr Gln Gly Gln Trp Thr Ile Gln Asn Arg Arg Leu Cys Gly Leu Arg 325 330 335 Ile Leu Gln Pro Tyr Ala Glu Arg Ile Pro Val Val Ala Thr Ala Gly 340 345 350 Ile Thr lie Asn Phe Thr Ser Gin lie Ser Leu Thr Gly Pro Gly Val 355 360 365 Arg Val His Tyr Gly Leu Tyr Asn Gin Ser Asp Pro Cys Pro Gly Glu 370 375 380 Phe Leu Cys Ser Val Asn Gly Leu Cys Val Pro Ala Cys Asp Gly Val 385 390 395 400 Lys Asp Cys Pro Asn Gly Leu Asp Glu Arg Asn Cys Val Cys Arg Ala 405 410 415 Thr Phe Gin Cys Lys Glu Asp Ser Thr Cys lie Ser Leu Pro Lys Val 420 425 430 Cys Asp Gly Gin Pro Asp Cys Leu Asn Gly Ser Asp Glu Glu Gin Cys 435 440 445 Gln Glu Gly Val Pro Cys Gly Thr Phe Thr Phe Gin Cys Glu Asp Arg 450 455 460 Ser Cys Val Lys Lys Pro Asn Pro Gin Cys Asp Gly Arg Pro Asp Cys 465 470 475 480 Arg Asp Gly Ser Asp Glu Glu His Cys Asp Cys Gly Leu Gin Gly Pro 485 490 495 Ser Ser Arg lie Val Gly Gly Ala Val Ser Ser Glu Gly Glu Trp Pro 500 505 510 Trp Gln Ala Ser Leu Gln Val Arg Gly Arg His Ile Cys Gly Gly Ala 515 520 525 Leu Ile Ala Asp Arg Trp Val Ile Thr Ala Ala His Cys Phe Gln Glu 530 535 540 Asp Ser Met Ala Ser Thr Val Leu Trp Thr Val Phe Leu Gly Lys Val 545 550 555 560 Trp Gln Asn Ser Arg Trp Pro Gly Glu Val Ser Phe Lys Val Ser Arg 565 570 575 Leu Leu Leu His Pro Tyr His Glu Glu Asp Ser His Asp Tyr Asp Val 580 585 590 Ala Leu Leu Gln Leu Asp His Pro Val Val Arg Ser Ala Ala Val Arg 595 600 605 Pro Val Cys Leu Pro Ala Arg Ser His Phe Phe Glu Pro Gly Leu His 610 615 620 Cys Trp Ile Thr Gly Trp Gly Ala Leu Arg Glu Gly Gly Pro Ile Ser 625 630 635 640 Asn Ala Leu Gln Lys Val Asp Val Gln Leu Ile Pro Gln Asp Leu Cys 645 650 655 Ser Glu Val Tyr Arg Tyr Gln Val Thr Pro Arg Met Leu Cys Ala Gly 660 665 670 Tyr Arg Lys Gly Lys Lys Asp Ala Cys Gin Gly Asp Ser Gly Gly Pro 675 680 685 Leu Val Cys Lys Ala Leu Ser Gly Arg Trp Phe Leu Ala Gly Leu Val 690 695 700 Ser Trp Gly Leu Gly Cys Gly Arg Pro Asn Tyr Phe Gly Val Tyr Thr 705 710 715 720 Arg lie Thr Gly Val lie Ser Trp lie Gin Gin Val Val Thr Gly Gly 725 730 735 Gly Gly Ser Gly Gly Gly Gly Ser His His His His His His Gly Ser 740 745 750 His His His His 755 <210> 21 <211> 521 <212> PRT <213> Artificial Sequence <220> <223> Human Proteinase-2 Masked ECD Protein his Tag <400> 21 Tyr Lys Ala Glu Val Met Val Ser Gin Val Tyr Ser Gly Ser Leu Arg 1 5 10 15 Val Leu Asn Arg His Phe Ser Gin Asp Leu Thr Arg Arg Glu Ser Ser 20 25 30 Ala Phe Arg Ser Glu Thr Ala Lys Ala Gln Lys Met Leu Lys Glu Leu 35 40 45 Ile Thr Ser Thr Arg Leu Gly Thr Tyr Tyr Asn Ser Ser Ser Val Tyr 50 55 60 Ser Phe Gly Glu Gly Pro Leu Thr Cys Phe Phe Trp Phe Ile Leu Gln 65 70 75 80 Ile Pro Glu His Arg Arg Leu Met Leu Ser Pro Glu Val Val Gln Ala 85 90 95 Leu Leu Val Glu Glu Leu Leu Ser Thr Val Asn Ser Ser Ala Ala Val 100 105 110 Pro Tyr Arg Ala Glu Tyr Glu Val Asp Pro Glu Gly Leu Val Ile Leu 115 120 125 Glu Ala Ser Val Lys Asp Ile Ala Ala Leu Asn Ser Thr Leu Gly Cys 130 135 140 Tyr Arg Tyr Ser Tyr Val Gly Gln Gly Gln Val Leu Arg Leu Lys Gly 145 150 155 160 Pro Asp His Leu Ala Ser Ser Cys Leu Trp His Leu Gln Gly Pro Lys 165 170 175 Asp Leu Met Leu Lys Leu Arg Leu Glu Trp Thr Leu Ala Glu Cys Arg 180 185 190 Asp Arg Leu Ala Met Tyr Asp Val Ala Gly Pro Leu Glu Lys Arg Leu 195 200 205 Ile Thr Ser Val Tyr Gly Cys Ser Arg Gln Glu Pro Val Val Glu Val 210 215 220 Leu Ala Ser Gly Ala Ile Met Ala Val Val Trp Lys Lys Gly Leu His 225 230 235 240 Ser Tyr Tyr Asp Pro Phe Val Leu Ser Val Gln Pro Val Val Phe Gln 245 250 255 Ala Cys Glu Val Asn Leu Thr Leu Asp Asn Arg Leu Asp Ser Gln Gly 260 26...

Claims

1. A binding polypeptide that binds to human MTP-2, comprising... (a) A heavy chain variable (VH) domain comprising heavy chain complementarity-determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, wherein HCDR1 is composed of the amino acid sequence of SEQ ID NO: 48, HCDR2 is composed of the amino acid sequence of SEQ ID NO: 49, and HCDR3 is composed of the amino acid sequence of SEQ ID NO: 50, and (b) A light chain variable (VL) domain comprising light chain complementarity-determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, wherein LCDR1 is composed of the amino acid sequence of SEQ ID NO: 58, LCDR2 is composed of the amino acid sequence of SEQ ID NO: 59, and LCDR3 is composed of the amino acid sequence of SEQ ID NO:

60. The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 regions are CDR sequences defined according to the IMGT method.

2. The binding polypeptide according to claim 1, wherein... (a) The VH domain contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 67, and (b) The VL domain contains an amino acid sequence that is at least 90% identical to SEQ ID NO:

63.

3. The binding polypeptide according to claim 2, wherein... (a) The VH domain contains the amino acid sequence of SEQ ID NO: 67, and (b) The VL domain contains an amino acid sequence containing SEQ ID NO:

63.

4. The binding polypeptide according to claim 3, wherein... (a) The VH domain consists of an amino acid sequence containing SEQ ID NO: 67, and (b) The VL domain consists of an amino acid sequence containing SEQ ID NO:

63.

5. The binding polypeptide of claim 1, wherein the binding polypeptide comprises an antibody constant region.

6. The binding polypeptide according to claim 5, wherein the binding polypeptide is an IgG antibody.

7. The binding polypeptide of claim 6, wherein the binding polypeptide comprises the human IgG4PE constant region.

8. The binding polypeptide according to claim 5, comprising... (a) A heavy chain comprising the amino acid sequence of SEQ ID NO: 69, and (b) A light chain comprising the amino acid sequence of SEQ ID NO:

65.

9. The binding polypeptide according to claim 5, comprising... (a) A heavy chain, said heavy chain consisting of the amino acid sequence of SEQ ID NO: 69, and (b) A light chain consisting of the amino acid sequence of SEQ ID NO:

65.

10. The binding polypeptide of claim 5, wherein the binding polypeptide comprises a YTE mutation.

11. A nucleic acid encoding the binding polypeptide according to claim 1.

12. An in vitro host cell comprising the nucleic acid according to claim 11.

13. A composition comprising a binding polypeptide according to any one of claims 1 to 10, formulated together with a pharmaceutically acceptable excipient.

14. A composition comprising (i) a binding polypeptide according to any one of claims 1-10, and (ii) a TGFβ superfamily ligand antagonist, wherein the TGFβ superfamily ligand is an activin IIB receptor Fc fusion protein.

15. A composition comprising (i) a binding polypeptide according to any one of claims 1-10, and (ii) erythropoietin.

16. Use of the composition according to claim 13 in the preparation of a medicament for reducing serum iron concentration in patients with β-thalassemia.

Images