Treatment for physiological iron overload

Abstract

To provide an agent for reducing iron overload in patients having conditions such as beta-thalassaemia and myelodysplastic syndrome (MDS).SOLUTION: Antibodies to enzyme matriptase-2 (MTP-2) are presented. Inhibiting 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 to a serine protease domain) can be used to treat iron overload, which is a feature of diseases such as beta-thalassaemia and which otherwise leads to toxic accumulation of iron. Combination of an MTP-2 inhibitor with an activin receptor ligand trap, or with erythropoeitin, provides additional therapeutic effects.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to agents that reduce iron overload in patients with conditions such as beta-thalassemia and myelodysplastic syndromes (MDS). [Background technology]

[0002] Iron is essential for erythropoiesis, the production of red blood cells that transport oxygen from the lungs to other tissues in the body. However, excess iron is toxic due to its ability to generate reactive oxygen species, and therefore, its absorption by the duodenum must be tightly regulated. The hepatic peptide hormone hepcidin plays a central role in matching iron absorption to the body's iron needs. Hepcidin negatively regulates cellular iron efflux by promoting the degradation of ferroportin, the only known iron exporter. Ferroportin is expressed in cells that are the major iron storage compartment, such as macrophages, and on the basolateral side of duodenal cells. Negative regulation of ferroportin by hepcidin therefore limits duodenal iron absorption and iron release from iron-storing cells such as macrophages. It is well established that activation of the BMP-SMAD signaling pathway in hepatocytes via secretion of BMP ligands (mainly 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 coreceptors such as HJV (Non-Patent Document 1), thereby reducing hepcidin expression and increasing dietary iron uptake and iron release from cellular stores. MTP-2 expression is induced by BMP6, which is essential for hepcidin expression, and by excess iron (Non-Patent Document 2). Thus, BMP6 stimulates hepcidin expression, but also increases the expression of negative regulators that maintain a negative feedback mechanism to prevent iron dysregulation (Non-Patent Document 3). Figure 1.

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

[0004] Beta-thalassemia is an inherited hemoglobinopathy caused by a genetic defect 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 contains a heme group, which reversibly binds oxygen to its central iron. In beta-thalassemia, incomplete production of hemoglobin leads to ineffective erythropoiesis and consequent anemia (a lack of oxygen-carrying red blood cells). Excessive production of erythropoietin (EPO), which is upregulated in response to anemia, and / or elevated levels of the erythroblast hormone erythroferon, which result from abnormally increased and ineffective erythropoiesis, have an inhibitory effect on the iron regulator hepcidin, increasing iron absorption from the gastrointestinal tract and iron release from internal stores, resulting in iron overload. Interestingly, it is iron overload, not anemia itself, that is thought to shorten life expectancy in beta-thalassemia. Increased iron availability increases transferrin saturation and heme production, which, along with a compensatory increase in alpha-globin expression, leads to the formation of hemispheres, as well as increased reactive oxygen stress and apoptosis of erythroid progenitor cells. This highly stimulated but ineffective erythropoiesis results in the non-survival of many mature red blood cells, resulting in the enlarged spleen, or splenomegaly, that is also characteristic of this disease.

[0005] There are three types of beta-thalassemia, classified according to the degree of reduction in beta-chain synthesis. The homozygous form, beta-thalassemia major, is the most severe form of congenital hemolytic anemia and is characterized by the absence or severe suppression of functional beta-chain synthesis. Patients require very frequent blood transfusions ("transfusion-dependent beta-thalassemia"). Patients with beta-thalassemia intermedia, on the other hand, are genetically heterozygous and do not require regular transfusions ("transfusion-independent beta-thalassemia"). There is a third form of beta-thalassemia, called beta-thalassemia minor, which is a mild, asymptomatic condition in which there is only moderate suppression of beta-chain synthesis.

[0006] An unfortunate consequence of transfusions in transfusion-dependent patients is that each transfusion contains at least 200 mg of iron, thus exacerbating the problems of iron overload and toxic tissue damage caused by untransferrin-bound iron, necessitating iron chelation therapy. Iron chelators can complex untransferrin-bound iron in the circulation, shifting the equilibrium and liberating iron from tissues to prevent tissue damage. However, chelators do not reduce transferrin saturation and therefore do not prevent increased hemilomic formation and apoptosis. Patients remain transfusion-dependent. Furthermore, 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, iron overload in some patients remains an unmet clinical need today.

[0007] Similarly, there is an unmet clinical need in the treatment of 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 resulting in one or more cytopenias and a variable tendency to develop acute myeloid leukemia (AML). Several forms of MDS, including MDS with chromosome deletion 5q (5q-MDS) and refractory anemia with ringed sideroblasts (RARS), are associated with anemia and toxic iron deposition within erythroid precursors. These forms of MDS are often associated with reduced hepcidin levels. While MDS can often be managed with regular transfusions, these transfusions can lead to secondary iron overload and reduced overall survival, as in beta-thalassemia. MDS patients are therefore typically treated with iron chelators once iron overload reaches a certain threshold.

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

[0009] Rare forms of anemia have also been shown to benefit from iron-reduction therapy in animal models. These anemias include Blackfan-Diamond anemia and sickle cell anemia. Iron deposition within the liver (primarily in parenchymal cells) has also been shown to promote oxidative stress and fibrosis in diseases such as hemochromatosis and hepatitis C infection, as well as in iron-loading anemia. 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 progresses to cirrhosis, accompanied by loss of liver function and progression to liver cancer.

[0010] There is a need for new medical treatments for patients with conditions such as those described above. Although overall survival can be improved by blood transfusions and iron chelation, these treatments do not address the underlying disease pathology and have the undesirable side effects described.

[0011] One area of ​​research involves investigating the biology of iron overload anemia, using mice with genetic beta-thalassemia as a model of human disease. Hbbth3 / + mice exhibit features similar to beta-thalassemia intermedia in humans, including Hb levels between 7 and 9 g / dL, abnormal red blood cell morphology, reticulocyte counts, ineffective and extramedullary erythropoiesis, hepatosplenomegaly, and iron overload in the liver and spleen—a complex phenotype that worsens with age (4).

[0012] Deletion of Tmprss6 (MTP2) or its reduction in expression has been shown to increase hepcidin expression and correct iron overload, splenomegaly, and anemia in Hbbth3 / + mice (Non-Patent Document 5; Non-Patent Document 6). Reducing Tmprss6 gene expression in an Hbbth3 / + mouse model of beta-thalassemia using lipid nanoparticle (LNP)-formulated Tmprss6 siRNA has been shown to induce hepcidin and attenuate tissue and serum iron levels. Furthermore, treatment of Hbbth3 / + animals with LNP-Tmprss6 siRNA improved red blood cell survival and erythropoiesis, thus substantially reducing anemia (Non-Patent Document 7).

[0013] Guo et al. demonstrated that Hbbth3 / + mice showed reduced formation of insoluble membrane-bound globin, reduced ROS, and reduced apoptosis, as well as reduced anemia, after treatment with antisense oligonucleotides against Tmprss6 (Guo et al., 2013, supra). These animals also showed reduced erythropoietin levels, significant improvement in ineffective erythropoiesis and splenomegaly, and increased total hemoglobin levels.

[0014] Consistent with these studies, knockout of Tmprss6 in the genetic background of thalassemia mice resulted in a significant reduction in iron overload and improved hemoglobin levels compared with Tmprss6+ thalassemia mice.

[0015] Gene therapy is a potential approach to address the underlying disease pathology in patients with iron overload disorders. In June 2019, the gene therapy Zynteglo received conditional regulatory approval from the EMA for the treatment of transfusion-dependent beta-thalassemia in patients aged 12 years and older who have no other treatment options. Gene therapy involves the ex vivo addition of a corrective beta-globin gene to the patient's bone marrow stem cells, which are then retransplanted into the patient. While potentially curative, this is a highly invasive and expensive procedure. As of November 2019, Zynteglo's approval has been delayed due to manufacturing issues, and it is not approved for the most severe type of beta-thalassemia (β0 / β0 genotype) because more than half of these patients experimentally treated with Zynteglo had to return to transfusions.

[0016] Fusion proteins containing the extracellular domain of the activin type II receptor linked to the Fc portion of human IgG1 are also under clinical investigation. These ligand traps act on the transforming growth factor β (TGFβ) superfamily to enhance late 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 to date, these proteins have not proven sufficient to achieve transfusion independence (Non-Patent Document 8).

[0017] Luspatercept (Rebrozil) was approved by the FDA on November 8, 2019, for the treatment of anemia in adults requiring regular blood transfusions in beta-thalassemia, based primarily on data provided by the Phase 3 BELIEVE trial. The study's primary clinical endpoints were the proportion of patients achieving at least a 33% reduction in transfusion burden from baseline and a reduction of at least 2 units from Weeks 13 to 24. Only 21.4% of patients achieved this, and only 7.6% to 10.3% achieved a transfusion reduction of more than 50% over the same time frame. Therefore, there remains an ongoing medical need for treatments in beta-thalassemia. [Prior art documents] [Non-patent literature]

[0018] [Non-Patent Document 1] Knutson, Nutrition Reviews 67:284~288, 2009 [Non-patent document 2] Meynard et al., Blood, 118:747-756, 2011 [Non-patent document 3] Wahedi et al., J Biol Chem, 292:18354~18371, 2017 [Non-patent document 4] Franceschi et al., Haematologica 91:1336–134, 2006 [Non-Patent Document 5] Guo et al., Journal of Clinical Investigation 123:1531–1541, 2013 [Non-patent document 6] Nai et al., Blood 119:5021-5029, 2012 [Non-Patent Document 7] Schmidt et al., Blood 121(7):1200-1208, 2013 [Non-patent document 8] Piga et al., Blood 133:1279-1289, 2019 Summary of the Invention [Problem to be solved by the invention]

[0019] Perhaps the simplest concept for increasing hepcidin levels through therapeutic intervention has been the therapeutic use of hepcidin, hepcidin derivatives, or analogs of hepcidin itself (Casu et al., Blood 128:265–276, 2016; Casu, Nemeth, and Rivera, Blood 131:1790–1794, 2018; Preza et al., Journal of Clinical Investigation 121:4880–4888, 2011). Despite their simple treatment concepts, none of these approaches have yet met the criteria for regulatory approval. At least two of these approaches have recently been terminated after clinical trials (La Jolla Pharmaceuticals' LJPC-401 in a Phase II trial for beta-thalassemia and Merganser's M-021 in a Phase I trial). However, there remains a significant medical need for the development of novel, effective treatments. [Means for solving the problem]

[0020] The present invention relates to binder polypeptides, such as antibodies, that bind to and inhibit MTP-2. The MTP-2 inhibitory binder polypeptides can be used to reduce iron overload in patients, including those with beta-thalassemia, MSD, and other iron overload anemias, as well as the additional conditions described herein. Various aspects of the invention relate to binder polypeptides, their use in the manufacture of medicaments and in methods of treating patients, methods of producing binder polypeptides, nucleic acids encoding binder polypeptides, and pharmaceutical formulations containing binder polypeptides.

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

[0022] Binder polypeptides according to the invention can be antibodies (e.g., IgG) or non-antibody molecules, such as alternative polypeptide scaffolds containing engineered binding loops. We describe antibodies and other binders that contain binding loops for MTP-2 (e.g., for the MTP-2 serine protease catalytic domain) that inhibit the enzymatic activity of MTP-2.

[0023] As embodiments, 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-1000, NORI-1010, NORI-1020, NORI-1030, NORI-1040, NORI-1050, NORI-1060, NORI-1070, NORI-1080, NORI-1090, NORI-1101, NORI-1112, NORI-1130, NORI-1140, NORI-1150, NORI-1160, NORI-1170, NORI-1181, NORI-1190, NORI-1202, NORI-1210, NORI-1211, NORI-1212, NORI-1213, NORI-1220, NORI-1221, NORI-1222, NORI-1230, NORI-1231, NORI-1232, NORI-1240, NORI-1243, NORI-1244, NORI-1245, NORI-1246, NORI-1247, NORI-1248, NORI-1250, NORI-1251, NORI-1252, NORI-1253, NORI-1254, NORI-1255, NORI-1256, NORI-1257, NORI-1258, NORI-1259 These include antibodies designated 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 through NORI-033"). These represent a diverse selection of antibody heavy and light chain sequences. All of these antibodies have been demonstrated to bind to MTP-2 and inhibit its catalytic activity. Selected exemplary antibodies have also been shown to successfully reduce hepcidin expression in vivo, which is a key biological process in physiological iron overload and demonstrates the potential of inhibitory anti-MTP-2 binder polypeptides to treat hematologic disorders and other conditions associated with iron overload.

[0024] A binder polypeptide according to the present invention may 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 may comprise an HCDR1, HCDR2, and / or HCDR3 that is the HCDR1, HCDR2, or HCDR3 of any of NORI-001 to NORI-033, and / or the antibody may comprise an LCDR1, LCDR2, or LCDR3 that is the LCDR1, LCDR2, or LCDR3 of any of NORI-001 to NORI-033. For example, the antibody may 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 may comprise a set of HCDRs that are the set of HCDRs of the VH domain of any of NORI-001 to NORI-033, and / or the antibody may comprise a set of LCDRs that are the set of LCDRs of the VL domain of any of NORI-001 to NORI-033. For example, the antibody may comprise 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, NORI-033, NORI-034, NORI-035, NORI-036, NORI-037, NORI-038, NORI-039, NORI-040, NORI-041, NORI-042, NORI-043, NORI-044, NORI-045, NORI-046, NORI-047, NORI-048, NORI-049, NORI-050, NORI-051, NORI-052, NORI-053, NORI-054, NORI-055, NORI-056, NORI-057, NORI-058, NORI-059, NORI-060 and / or NORI-031, NORI-032, NORI-033, NORI-034, NORI-035, NORI-036, NORI-037, NORI-038, NORI-039, NORI-040, NORI-041, NORI-042, NORI-043, NORI-044, NORI-045, NORI-046, NORI-047, NORI-048, NORI-049, NORI-050, NORI-051, NORI-052, NORI-053, NORI-054, NORI-055, NORI-056, NORI-057, NORI-058, NORI-059, NORI-060, NORI-061, NORI-062, NORI-063, NORI-064, NORI-065, NORI-066, NORI-067, NORI-068, NORI-069, NORI-070, NORI-071, NORI-072, NORI-073, NORI-074, NORI-075, NORI-076, NORI-077, NORI-078, NORI-079, NORI-080, NORI-081, NORI-082, NORI-083, NORI-084, NORI-085, NORI-086, NORI-087, NORI-088, NORI-089, NORI-090, NORI-101, NORI-102, NORI-103, NORI-104, NORI-105, NORI-106

[0027] The binder polypeptide may comprise a VH domain having at least 90% amino acid sequence identity with the VH domain of any of NORI-001 to NORI-033, and / or the binder polypeptide may comprise a VL domain having at least 90% amino acid sequence identity with the VL domain of any of NORI-001 to NORI-033. For example, the binder polypeptide may be any 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, NORI-033, NORI-034, NORI-035, NORI-036, NORI-037, NORI-038, NORI-039, NORI-040, NORI-041, NORI-042, NORI-043, NORI-044, NORI-045, NORI-046, NORI-047, NORI-048, NORI-049, NORI-050, NORI-051, NORI-052, NORI-053, NORI-054, NORI-055, NORI-056, NORI-057, NORI-058, NORI-059, NO The VH and VL domains may comprise the VH and VL domains of ORI-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 variant VH and / or VL domains having 90% sequence identity to said VH and / or VL domains.

[0028] In a first configuration, the antibody comprises: a VH domain comprising the NORI-003 HCDRs and having at least 90% sequence identity with the VH domain of NORI-003; and a VL domain comprising the NORI-003 LCDR and having at least 90% sequence identity with the VL domain of NORI-003 Includes.

[0029] The antibody may comprise a NORI-003 VH domain and a NORI-003 VL domain. Optionally, the antibody is an IgG comprising a NORI-003 heavy chain and a NORI-003 light chain.

[0030] In a second configuration, the antibody comprises: a VH domain comprising the NORI-006 HCDRs and having at least 90% sequence identity with the VH domain of NORI-006; and a VL domain comprising the NORI-006 LCDR and having at least 90% sequence identity with the VL domain of NORI-006 Includes.

[0031] The antibody may 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.

[0032] In a third configuration, the antibody comprises: a VH domain comprising the NORI-008 HCDRs and having at least 90% sequence identity with the VH domain of NORI-008; and A VL domain comprising the NORI-008 LCDR and having at least 90% sequence identity with the VL domain of NORI-008. Includes.

[0033] The antibody may 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.

[0034] In a fourth configuration, the antibody comprises: a VH domain comprising the NORI-011 HCDRs and having at least 90% sequence identity with the VH domain of NORI-011; and a VL domain comprising the NORI-011 LCDR and having at least 90% sequence identity with the VL domain of NORI-011 Includes.

[0035] The antibody may 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.

[0036] In various embodiments of the invention, the VH and / or VL domains optionally have a % sequence identity of greater than 90%, for example the % sequence identity may be 95% or greater, 98% or greater, or 99% or greater.

[0037] A binder polypeptide may comprise an antibody VH domain produced by recombination of v, d, and j gene segments, which are the v, d, and j gene segments from which the VH domain of any of NORI-001 to NORI-033 was produced. A binder polypeptide may comprise an antibody VL domain produced by recombination of v and j gene segments, which are the v and j gene segments from which the VL domain of any of NORI-001 to NORI-033 was produced. For example, a binder polypeptide may comprise a VH domain produced by recombination of v, d, and j gene segments from which the VH domain of any of NORI-001 to NORI-003 was produced, and a binder polypeptide may comprise an antibody VL domain produced by recombination of v and j gene segments, which are the v and j gene segments from which the VL domain of said antibody was produced.

[0038] Inhibition of MTP-2 enzymatic activity can be determined in an in vitro assay for inhibition of serine protease cleavage of an MTP-2 substrate to produce a detectable product. Such an in vitro enzyme assay can involve contacting a polypeptide with MTP-2 or the MTP-2 extracellular domain and detecting the degree of reduction in the production of a detectable product compared to a control assay lacking the polypeptide (a negative control polypeptide is included instead). The enzyme assay can be performed at various concentrations of the polypeptide to generate a dose-response curve from which an IC50 value can be calculated. Thus, inhibitors according to the present invention can be identified via their dose-dependent inhibition in such an enzyme assay.

[0039] 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 μM. A typical substrate is Boc-Gln-Gly-Arg-AMC, currently available as Baychem 4016429. MTP-2 in such an assay may have an activity rate of 0.075 U / μl. MTP-2 is provided as a purified protein in solution, e.g., the MTP-2 extracellular domain. Thus, the IC50 of the binder polypeptide is determined by the activity rate of purified MTP-2 extracellular domain in the presence of 50 μM Boc-Gln-Gly-Arg-AMC fluorescent substrate at 0.075 U / μl. This can be determined in an enzyme assay using the ectodomain. The binder polypeptide may have an IC50 of less than 100 nM in such an assay.

[0040] Alternatively to purified ECD, inhibition may be assayed in vitro using cell surface expressed MTP-2 in a cell-based assay using, for example, HEK293 cells.

[0041] The effect of MTP-2 inhibition (eg, reduction in expression of hepcidin, measurable as a reduction in mRNA for its encoding gene, hamp) is further detected in vivo to confirm inhibitory activity and biological relevance.

[0042] In various embodiments, the potency of an inhibitor is quantified according to its IC50 as measured in an in vitro assay for inhibition of MTP-2 enzymatic activity (e.g., an assay as described above). Preferably, a binder polypeptide according to the invention has an IC50 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 in an in vitro assay for inhibition of MTP-2 enzymatic activity. 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.

[0043] The potency is compared with one or more anti-MTP-2 antibodies described herein for reference. For example, an antibody comprising the VH and VL domains of any of NORI-001 to NORI-033 can be used as a reference antibody. The reference antibody is provided as an IgG. For example, a binder polypeptide according to the present invention may have an IC50 within 25% or 10% of the IC50 of any of NORI-001 to NORI-033, such as NORI-003 IgG, NORI-006 IgG, NORI-008 IgG, NORI-009 IgG, or NORI-011 IgG, or a binder polypeptide according to the present invention may have an IC50 lower than that of the reference antibody. "Within x% of" means that the IC50 of the test binder polypeptide is x% or less higher and x% or less than that of the reference antibody.

[0044] The inhibitory potency is compared to that of aprotinin, a 6500 dalton pan-serine protease inhibitor known to occupy the active site of serine proteases. The IC50 of the binder polypeptide is comparable to or lower than that of aprotinin. The binder polypeptide may have an IC50 within 50%, within 25%, or within 10% of that of aprotinin.

[0045] 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. Comparison of the inhibitory potency of binder polypeptides in the same enzyme assay using MTP-2 from different species provides an indication of the interspecies cross-reactivity of the binder polypeptide. In general, interspecies cross-reactivity is desirable because it allows the possibility of testing binder polypeptides in vivo in multiple species. For example, preclinical studies in laboratory animals (e.g., mouse, rat, or cynomolgus monkey) can be conducted before clinical studies in the target species (e.g., human). Preferably, binder polypeptides in accordance with the invention are cross-reactive for binding and inhibition of MTP-2 from multiple species. Preferably, binder polypeptides in accordance with the invention bind to and inhibit human and mouse MTP-2. More preferably, binder polypeptides in accordance with the invention bind to and inhibit human, mouse, rat, and cynomolgus monkey MTP-2.

[0046] A binder polypeptide may have an IC50 in an in vitro assay for inhibition of non-human (e.g., mouse, rat, and / or cynomolgus monkey) MTP-2 enzyme activity that is within 50%, within 25%, or within 20% of its IC50 in an in vitro assay for inhibition of human MTP-2 enzyme activity.

[0047] A binder polypeptide may have an IC50 in an in vitro assay for inhibition of non-human (e.g., mouse, rat, and / or cynomolgus monkey) MTP-2 enzyme activity that differs less than 100-fold, less than 50-fold, less than 10-fold, less than 5-fold, or less than 2-fold from its IC50 in an in vitro assay for inhibition of human MTP-2 enzyme activity.

[0048] Similarly, another measure of interspecies cross-reactivity is provided by comparing the affinity of a binder polypeptide for MTP-2 of one species with its affinity for MTP-2 of another species. Binding affinity (K), e.g., as determined by surface plasmon resonance, can be used to measure binding affinity (K). D ) are compared. The K of binding polypeptides to non-human (e.g., mouse, rat, and / or cynomolgus monkey) MTP-2 is D is its K binding to human MTP-2 D differs from less than 50-fold, less than 10-fold, less than 5-fold, or less than 2-fold.

[0049] The ability of a binder polypeptide to compete with a reference molecule for binding to MTP-2 is determined in vitro. Competition for binding to MTP-2 is 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 according to the region of MTP-2 to which the reference molecule binds. For example, a binder polypeptide according to the present invention can compete with aprotinin for binding to MTP-2. A binder polypeptide according to the present invention can compete 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 to NORI-033 is used as a reference antibody. The reference antibody is provided as an IgG. For example, in various embodiments, the binder polypeptide is selected from NORI-003 IgG, NORI-006 IgG, NORI-008 IgG, or NORI-011 IgG for binding to MTP-2. Alternatively, the reference antibody may be provided as an scFv. For example, in various embodiments, the binder polypeptide may be selected from the group consisting of NORI-003 scFv, NORI-006 scFv, NORI-008 scFv, and NORI-009 scFv for binding to MTP-2. scFv, or one that competes with NORI-011 IgG.

[0050] The IC50 can be determined in a competitive assay. For example, the binder polypeptide may have an IC50 of less than 20 nM in a competitive assay with labeled aprotinin for binding to human MTP-2. The mature aprotinin sequence of 58 amino acids shown in Table S can be used in the competitive assay.

[0051] Nucleic acids encoding the binder polypeptides described herein are also provided, as are cells containing the nucleic acid. An in vitro host cell can contain the nucleic acid, which is optionally integrated into the cellular (e.g., genomic) DNA of the host cell or transiently transfected (e.g., plasmid DNA).

[0052] These and other aspects and embodiments of the invention, including methods of producing binder polypeptides, pharmaceutical compositions, and methods of treating patients, are described in more detail below.

[0053] item Antibodies against the enzyme matriptase-2 (MTP-2) are presented. Inhibition of MTP-2 reduces dietary iron uptake and reduces iron release from the body's cellular stores. Inhibitors of MTP-2 (such as antibodies against the serine protease domain) can be used to treat iron overload, a hallmark of diseases such as beta-thalassemia and other conditions that result in toxic accumulation of iron. Combining MTP-2 inhibitors with activin receptor ligand traps or with erythropoietin provides additional therapeutic benefits.

[0054] The following numbered items represent embodiments of the present invention and are part of the description:

[0055] 1. An isolated binder polypeptide that binds to and inhibits the enzymatic activity of MTP-2, optionally binding to the serine protease catalytic domain of MTP-2.

[0056] 2. The binder polypeptide according to item 1, comprising an immunoglobulin domain in which the binding site for MTP-2 is formed by a loop region of the immunoglobulin domain.

[0057] 3. The binder polypeptide according to item 2, which is an antibody, optionally a human antibody.

[0058] 4. The binder polypeptide according to any one of items 1 to 3, wherein the MTP-2 is human MTP-2.

[0059] 5. The binder polypeptide according to item 4, wherein the MTP-2 is human MTP-2 and mouse MTP-2.

[0060] 6. The binder polypeptide of item 4 or 5, which binds to human MTP-2 containing a sequence polymorphism in which residue 253 is K or E and residue 736 is V or A.

[0061] 7. A binder polypeptide according to any one of items 1 to 6, which does not bind to MTP-1 and optionally does not bind to other members of the type II transmembrane serine protease family.

[0062] 8. The binder polypeptide according to any one of items 1 to 7, which exhibits dose-dependent inhibition of MTP-2 serine protease activity in an enzymatic assay using the MTP-2 extracellular domain and a fluorescent MTP-2 substrate at a final concentration of 50 μM.

[0063] 9. The binder polypeptide according to item 8, having an IC50 of less than 100 nM in an enzyme assay against human MTP-2 extracellular domain and a fluorescent MTP-substrate at a final concentration of 50 μM.

[0064] 10. The binder polypeptide according to item 8 or 9, having an IC50 of less than 100 nM in an enzyme assay against mouse MTP-2 extracellular domain and a fluorescent MTP-substrate at a final concentration of 50 μM.

[0065] 11. The binder polypeptide according to item 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 said assay using the human MTP-2 extracellular domain.

[0066] 12. The binder polypeptide according to any one of items 1 to 11, which exhibits dose-dependent inhibition of MTP-2 serine protease activity in an enzymatic assay using human MTP-2 expressed on the surface of HEK293 cells and a fluorescent MTP-2 substrate at a final concentration of 50 μM.

[0067] 13. A binder polypeptide according to item 11, having an IC50 of less than 100 nM in an enzyme assay using human MTP-2 expressed on the surface of HEK293 cells and a fluorescent MTP-2 substrate at a final concentration of 50 μM.

[0068] 14. A binder polypeptide according to any one of items 1 to 13, 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.

[0069] 15. The binder polypeptide of item 14, which competes with an IgG comprising the VH and VL domains of NORI-003, NORI-006, NORI-008, or NORI-011 for binding to the serine protease catalytic domain of human and / or mouse MTP-2.

[0070] 16. A binder polypeptide according to any one of items 1 to 15, which competes with aprotinin for binding to the serine protease catalytic domain of human and / or mouse MTP-2.

[0071] 17. The binder polypeptide according to item 16, having an IC50 of less than 100 nM in a competition assay with labeled aprotinin for binding to human and / or mouse MTP-2.

[0072] 18. The binder polypeptide according to item 17, having an IC50 of less than 50 nM in a competition assay with labeled aprotinin for binding to human and / or mouse MTP-2.

[0073] 19. The binder polypeptide according to item 18, which has an IC50 of less than 20 nM in a competition assay with labeled aprotinin for binding to human MTP-2.

[0074] 20. The binder polypeptide according to any one of items 1 to 19, which has an affinity (Kd) for human MTP-2 of less than 50 nM as determined by surface plasmon resonance.

[0075] 21. The binder polypeptide according to any one of items 1 to 20, which has an affinity (Kd) for mouse MTP-2 of less than 50 nM as determined by surface plasmon resonance.

[0076] 22. The binder polypeptide according to item 21, wherein the Kd for mouse MTP-2 is within 50-fold of the Kd for human MTP-2.

[0077] 23. The binder polypeptide according to any of items 1 to 22, comprising an antibody heavy chain variable (VH) domain obtained by recombination of a set of germline vdj gene segments as set forth in Table G for any of NORI-001 to NORI-033, and / or an antibody light chain variable (VL) domain obtained by recombination of a set of germline vj gene segments as set forth in Table G for any of NORI-001 to NORI-033.

[0078] 24. The VH and VL domains are 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, 24. The binder polypeptide of item 23, obtained by recombination of a set of germline gene segments set forth in Table G for 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.

[0079] 25. Germline vdj gene segment: IGHV3-9 * 01, IGHD4-17 * 01, and IGHJ6 * 02. IGHV4-61 * 01, IGHD3-22 * 01, and IGHJ5 * 02. IGHV3-49 * 05, IGHD3-9 * 01, and IGHJ4 * 02, or IGHV3-13 * 01, IGHD3-10 * 01, and IGHJ3 * 02 25. The binder polypeptide according to any one of items 1 to 24, comprising an antibody heavy chain variable (VH) domain obtained by recombination of

[0080] 26. Germline vj gene segment: IGLV2-8 * 01 and IGLJ2 * 01. IGKV1D-33 * 01 and IGKJ5 * 01. IGKV1D-33 * 01 and IGKJ4 * 01, and IGKV3D-7 * 01 and IGKJ1 * 01 26. The binder polypeptide according to any one of items 1 to 25, comprising an antibody light chain variable (VL) domain obtained by recombination of

[0081] 27. The binder polypeptide according to any of items 1 to 26, wherein the binder polypeptide comprises a VH domain comprising a pair of heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and a VL domain comprising a pair of light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, wherein the pair of HCDRs is a pair of HCDRs from any of NORI-001 to NORI-033, and / or the pair of LCDRs is a pair of LCDRs from any of NORI-001 to NORI-033.

[0082] 28. A binder polypeptide according to item 27, wherein the set of HCDRs is the set of CDRs of NORI-003 and the set of LCDRs is the set of LCDRs of NORI-003.

[0083] 29. A binder polypeptide according to item 27, wherein the set of HCDRs is the set of CDRs of NORI-006 and the set of LCDRs is the set of LCDRs of NORI-006.

[0084] 30. A binder polypeptide according to item 27, wherein the set of HCDRs is the set of CDRs of NORI-011 and the set of LCDRs is the set of LCDRs of NORI-011.

[0085] 31. A binder polypeptide according to item 27, wherein the set of HCDRs is the set of CDRs of NORI-008 and the set of LCDRs is the set of LCDRs of NORI-008.

[0086] 32. The binder polypeptide according to any one of items 1 to 31, comprising a VH domain having at least 90% amino acid sequence identity with 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 with the VL domain of any one of NORI-001 to NORI-033.

[0087] 33. A VH domain comprising the NORI-003 HCDR and having at least 90% amino acid sequence identity with the VH domain of NORI-003; and A VL domain containing the NORI-003 LCDR and having at least 90% amino acid sequence identity with the VL domain of NORI-003. 33. The binder polypeptide according to item 32, comprising:

[0088] 34. A binder polypeptide according to item 33, comprising the NORI-003 antibody VH domain and the NORI-003 VL domain.

[0089] 35. A VH domain comprising the NORI-006 HCDR and having at least 90% amino acid sequence identity with the VH domain of NORI-006; and A VL domain comprising the NORI-006 LCDR and having at least 90% amino acid sequence identity with the VL domain of NORI-006. 33. The binder polypeptide according to item 32, comprising:

[0090] 36. A binder polypeptide according to item 32, comprising the NORI-011 VH domain and the NORI-011 VL domain.

[0091] 37. A VH domain comprising the NORI-011 HCDR and having at least 90% amino acid sequence identity with the VH domain of NORI-011; and A VL domain containing the NORI-011 LCDR and having at least 90% amino acid sequence identity with the VL domain of NORI-011. 37. The binder polypeptide according to item 36, comprising:

[0092] 38. A binder polypeptide according to item 32, comprising a NORI-008 VH domain and a NORI-008 VL domain.

[0093] 39. A VH domain comprising the NORI-008 HCDR and having at least 90% amino acid sequence identity with the VH domain of NORI-008; and A VL domain containing the NORI-008 LCDR and having at least 90% amino acid sequence identity with the VL domain of NORI-008. 39. The binder polypeptide according to item 38, comprising:

[0094] 38. A binder polypeptide according to item 37, comprising a NORI-008 VH domain and a NORI-008 VL domain.

[0095] 39. The binder polypeptide according to any of items 1 to 38, comprising an antibody constant region.

[0096] 40. The binder polypeptide according to item 39, which is an IgG antibody.

[0097] 41. The binder polypeptide according to item 40, comprising a human IgG4PE constant region.

[0098] 42. The binder polypeptide according to item 41, comprising the NORI-003 antibody heavy chain and the NORI-003 antibody light chain.

[0099] 43. The binder polypeptide according to item 41, comprising the NORI-006 antibody heavy chain and the NORI-006 antibody light chain.

[0100] 44. The binder polypeptide according to item 41, comprising a NORI-011 antibody heavy chain and a NORI-011 antibody light chain.

[0101] 45. The binder polypeptide according to item 41, comprising a NORI-008 antibody heavy chain and a NORI-008 antibody light chain.

[0102] 46. ​​An isolated antibody comprising a VH domain and a VL domain according to any one of items 23 to 38.

[0103] 47. A VH domain and a VL domain, which are the VH and VL domains of any of NORI-001 to NORI-033; or comprising said VH and VL domains, wherein one or more non-germline residues in the framework regions have reverted to germline. Isolated antibodies.

[0104] 48. The antibody of item 47, comprising the VH and VL domains of NORI-003, or said VH and VL domains in which one or more non-germline residues in the framework regions have been reverted to germline.

[0105] 49. The antibody of item 47, comprising the VH and VL domains of NORI-006, or said VH and VL domains in which one or more non-germline residues in the framework regions have been reverted to germline.

[0106] 50. The antibody according to item 47, comprising the VH and VL domains of NORI-011, or said VH and VL domains in which one or more non-germline residues in the framework regions have been reverted to germline.

[0107] 51. The antibody according to item 47, comprising the VH and VL domains of NORI-008, or said VH and VL domains in which one or more non-germline residues in the framework regions have been reverted to germline.

[0108] 52. An isolated antibody comprising the heavy chain and light chain of any of NORI-001 to NORI-033.

[0109] 53. NORI-003 antibody heavy chain and NORI-003 antibody light chain, NORI-006 antibody heavy chain and NORI-006 antibody light chain, a NORI-011 antibody heavy chain and a NORI-011 antibody light chain, or NORI-008 antibody heavy chain and NORI-008 antibody light chain A monoclonal IgG antibody comprising:

[0110] 54. A nucleic acid encoding the binder polypeptide according to any one of items 1 to 45 or the antibody according to item 46 or item 53.

[0111] 55. An in vitro host cell containing the nucleic acid according to item 54.

[0112] 56. A composition comprising a binder polypeptide according to any one of items 1 to 45 or an antibody according to any one of items 46 to 53, formulated with a pharmaceutically acceptable excipient.

[0113] 57. The composition according to item 56, for subcutaneous administration.

[0114] 58. A composition comprising the nucleic acid according to item 54 for in vivo gene therapy.

[0115] 59. Items 56-5 for use in the treatment of the human or animal body by therapy 8. The composition according to any one of claims 1 to 8.

[0116] 60. A drug combination comprising (i) an inhibitor of MTP-2, and (ii) an antagonist of a TGFβ superfamily ligand.

[0117] 61. The combination according to item 60 for use in treating iron overload and normalizing erythropoiesis in a patient.

[0118] 62. A method for treating iron overload and normalizing erythropoiesis in a patient, the method comprising administering to the patient (i) an inhibitor of MTP-2, and (ii) an antagonist of a TGFβ family ligand, wherein (i) and (ii) are administered simultaneously or sequentially.

[0119] 63. The combination according to item 61 or the method according to item 62, wherein the patient has beta thalassemia (optionally beta thalassemia major), MDS, Blackfan-Diamond anemia, hemochromatosis type 1, or hemochromatosis type 3.

[0120] 64. The combination or method according to any of items 60 to 63, wherein the antagonist is an activin II receptor ligand trap.

[0121] 65. The combination or method according to item 64, wherein the antagonist is an activin IIB receptor Fc fusion protein.

[0122] 66. The combination or method according to item 65, wherein the antagonist is luspatercept.

[0123] 67. The combination or method according to item 64, wherein the antagonist is an activin IIA receptor Fc fusion protein.

[0124] 68. The combination or method according to item 67, wherein the antagonist is sotatercept.

[0125] 69. A drug combination comprising (i) an inhibitor of MTP-2, and (ii) erythropoietin.

[0126] 70. The combination according to item 69 for use in treating anemia associated with iron overload in a patient.

[0127] 71. A method for treating anemia associated with iron overload in a patient, the method comprising administering to the patient (i) an inhibitor of MTP-2, and (ii) erythropoietin, wherein (i) and (ii) are administered simultaneously or sequentially.

[0128] 72. The combination or method according to any of items 1 to 71, wherein the inhibitor of MTP-2 is a binder polypeptide or antibody according to any of items 1 to 71.

[0129] 72. In patients Reduces the absorption of dietary iron Treating iron overload, Increases hepcidin expression from liver cells Reduces anemia caused by iron overload, Decreasing serum iron levels, and / or Decreases iron saturation in transferrin A method comprising the step of administering to a patient the composition according to any one of items 56 to 58.

[0130] 73. The method of item 72, wherein the patient has beta-thalassemia (e.g., beta-thalassemia major or intermedia), 5q-MDS, or RARS.

[0131] 74. The method of item 72 or item 73, further comprising administering to the patient an antagonist of a TGFβ family ligand.

[0132] 75. The method according to item 74, wherein the antagonist is an activin II receptor ligand trap.

[0133] 76. The method according to item 75, wherein the antagonist is an activin IIB receptor Fc fusion protein.

[0134] 77. The method according to item 76, wherein the antagonist is luspatercept.

[0135] 78. The method of item 75, wherein the antagonist is an activin IIA receptor Fc fusion protein.

[0136] 79. The method according to item 76, wherein the antagonist is sotatercept.

[0137] 80. The method of item 72 or item 73, wherein the method further comprises administering an erythropoiesis stimulating agent to the patient, optionally wherein the erythropoiesis stimulating agent is erythropoietin.

[0138] 81. The method of item 72 or item 73, comprising administering to the patient a further therapeutic agent, for example luspatercept, to reduce iron overload.

[0139] 82. The composition according to any one of items 56 to 58 for use in the method according to any one of items 72 to 81.

[0140] 83. Use of a composition according to any one of items 56 to 58 for the manufacture of a medicament for the treatment of a patient, comprising the method according to any one of items 72 to 81.

[0141] 84. In patients Reduces the absorption of dietary iron Treating iron overload, Increases hepcidin expression from liver cells Reduces anemia caused by iron overload, Decreasing serum iron levels, and / or Decreases iron saturation in transferrin 59. A therapeutic agent for reducing iron overload for use in a method, the method comprising the step of administering to a patient the therapeutic agent and the composition according to any one of items 56 to 58.

[0142] 85. A therapeutic agent comprising erythropoietin (EPO) for use in a method for stimulating erythropoiesis in a patient, the method comprising administering to the patient the therapeutic agent and a composition according to any of items 56 to 58.

[0143] 86. A method for promoting red blood cell maturation in a patient, comprising administering to a subject a therapeutically effective amount of TGF-β agonist. 59. A therapeutic agent comprising an antagonist of a myristate ligand, the method comprising administering to a patient the therapeutic agent and the composition according to any one of items 56 to 58.

[0144] 87. The method according to any one of items 74 to 81, the composition for use according to item 82, the use of the composition according to item 83, or the therapeutic agent for use according to any one of items 84 to 86, wherein the method comprises a step of separately and sequentially administering the therapeutic agent and the composition to a patient.

[0145] Matriptase-2 (MTP-2) MTP-2 is a type II transmembrane trypsin-like serine protease that belongs to the type II transmembrane serine protease (TTSP) family. The corresponding gene for MTP-2, TMPRSS6, is located on 22q12.3.

[0146] The canonical isoform of MTP-2, isoform 1, is an 811-amino acid protein with a molecular mass of 90 kDa. It has a conserved structure similar to closely related TTSP family members such as matriptase-1 and enteropeptidase. It consists of a small N-terminal intracellular signal peptide that serves as a signal anchor and a single transmembrane domain, followed by a sea urchin sperm protein, enteropeptidase, and agrin (SEA) domain, two complement factor C1r / C1s, sea urchin embryonic growth factor, and bone morphogenetic protein (CUB) domains, and a stem region containing three low-density lipoprotein receptor (LDLR) class A repeats. Finally, a C-terminal serine protease (SP) domain contains the highly conserved catalytic triad of histidine (617), aspartic acid (668), and serine (762), which are required for enzymatic function. (Figure 2)

[0147] Like other TTSP members, the correct processing of MTP-2 into its membrane-bound, enzymatically active form is a complex process in which multiple disulfide bonds within the protein and the presence of additional endogenous proteins play important roles. MTP-2 is synthesized within the endoplasmic reticulum membrane and transported to the cell surface as an inactive zymogen, whereupon MTP-2 undergoes autocleavage 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 importantly connects the domain to the membrane-bound backbone.

[0148] The cleaved form of MTP-2 represents the active form and remains largely membrane-bound, but in this case, MTP-2 can cleave other membrane-bound targets on the cell surface. However, overexpression of MTP-2 in vitro has led to the discovery of an "effluxed" active form of MTP-2 found in the supernatant of cultured cells. It remains unknown whether this effluxed form is part of the native MTP-2 biology with in vivo function or simply a result of overexpression in a cell-based system.

[0149] The amino acid sequence of MTP-2 is shown in Table S. For example, human MTP-2 is It has the amino acid sequence of ID Q8IU80, including an N-terminal leader sequence and cytoplasmic domain, transmembrane domain, and extracellular domain (ECD). The ECD comprises amino acids 84-811 of the full-length protein. MTP-2 fragments containing the ECD, amino acids 78-811, can be recombinantly produced and used in the assays described herein (e.g., His-tagged forms). Production of MTP-2 for use in the assays is detailed in Example 5.

[0150] Binder polypeptides according to the present invention include cell surface expressed MTP-2, isolated MTP-2, and The soluble MTP-2 ECD can bind to and inhibit any one or more or all of the shed soluble MTP-2 ECD and the shed soluble MTP-2 ECD.

[0151] The MTP-2 referred to herein may be human or non-human (e.g., mouse, rat, or cynomolgus monkey) unless the context dictates otherwise. Preferably, the MTP-2 is human MTP-2.

[0152] Because MTP-2 processing is complex and involves a large portion of the protein structure, mutations in the protein can result in loss of function. Additionally, TMPRSS6 polymorphisms, such as rs855791, are known to result in increased MTP-2 activity and more efficient inhibition of hepcidin. The R576A mutation mutates a critical arginine required for SP domain cleavage and full activation of MTP-2, thus maintaining the protein as an inactive zymogen. The S762A mutation mutates a critical serine residue within the catalytic triad of the SP domain, completely abolishing activity. Thus, because autoactivation is required for autocleavage, the protein remains an inactive zymogen. The E114K mutation has been described in patients with nonfunctional MTP-2 expression. This mutation resides within the SEA domain and therefore most likely prevents proper delivery of the protein to the cell surface.

[0153] Four variants of MTP-2 are known, covering approximately 92% of the human population: isoform 1 (27.4%); rs855791 SNP (27.2%), resulting in a single amino acid change from valine to alanine at position 736; variants with both V736A and an additional K253E mutation (25.9%); and K253E variant alone (11.4%).

[0154] The binder polypeptides preferably bind to all four such variants and are therefore suitable for treating all or a large proportion of the human population by inhibiting the MTP-2 variants expressed by that majority. Thus, the binder polypeptides can bind to human MTP-2 containing a sequence polymorphism in which residue 253 is K or E and residue 736 is V or A.

[0155] Four isoforms of MTP-2 are known. Isoform 1, known as the standard isoform, is 811 amino acids long and is primarily expressed in the testes. Isoform 2, the predominant isoform in the liver, lacks the N-terminal 9 intracellular amino acids (802 amino acids). This N-terminal region is thought to be involved in the internalization of membrane-bound MTP-2 and therefore internalizes more slowly than isoform 1. Isoform 3, along with isoform 1, is also primarily expressed in the testes. It utilizes an alternative splicing variant of exon 10, which promotes the expression of a truncated form that has the 9 N-terminal amino acids but lacks the SP domain and is therefore functionally inactive. Isoform 4 has the same exons as isoform 2, but also contains an additional 22 amino acid exon between exons 16 and 17 that disrupts the function of the SP domain and is also functionally inactive. Isoform 4 is thought to be expressed in tissues that also express isoform 2, and because isoforms 3 and 4 have lost their function, it is thought to be a dominant-negative regulator of isoforms 1 and 2. It has been shown that expression of isoforms 3 or 4 can block isoform 2-mediated cleavage of HJV.

[0156] The binder polypeptide may bind to at least active isoforms 1 and 2. Optionally, the binder polypeptide may bind to isoform 3. Optionally, the binder polypeptide may bind to isoform 4. Optionally, the binder polypeptide does not bind to isoform 3. Optionally, the binder polypeptide does not bind to isoform 4. Lack of binding to inactive isoforms may be advantageous for therapeutic molecules aimed at inhibiting the activity of MTP-2. Binding to the serine protease catalytic domain of isoform 1 and / or isoform 2, and lack of binding to isoform 3 and / or isoform 4, may be advantageous.

[0157] Binding to MTP-2 As mentioned above, MTP-2 is a multidomain protein, and various mutations in the protein have been shown to result in loss of function. Therefore, binder polypeptides that recognize binding sites in various domains and inhibit the enzymatic activity of the protein can be generated.

[0158] The binder polypeptide may bind to the serine protease catalytic domain of MTP-2. The binder polypeptide may bind to autoactivated MTP-2. The binder polypeptide may bind to the MTP-2 ECD. The binder polypeptide may bind to the MTP-2 zymogen.

[0159] Binder polypeptides that bind to the serine protease catalytic domain are identified as those that bind to MTP-2 containing the domain but not to MTP-2 lacking the domain. A "headless" variant of MTP-2 can be constructed with a C-terminal truncation that deletes the serine protease domain but still contains the remaining ECD. Binding to the full-length MTP-2 ECD and the absence of binding to the headless MTP-2 ECD indicates that the binder polypeptide recognizes an epitope within the serine protease catalytic domain. Binding can be measured by HTRF assays or by surface plasmon resonance, exemplary protocols for which are provided herein. Binding to the serine protease catalytic domain can also be identified in enzyme inhibition assays using MTP-2 ECD and headless MTP-2 ECD. Binders that bind to the serine protease catalytic domain may exhibit dose-dependent binding to MTP-2 ECD in such assays, and do not exhibit dose-dependent binding to headless MTP-2 ECD in said assays.

[0160] Binding to cell surface expressed MTP-2 (eg, expressed on HEK293 cells) can be detected by fluorescence activated cell sorting (FACS).

[0161] The binder polypeptide can compete with aprotinin for binding to MTP-2 (eg, for the MTP-2 ECD, eg, its serine protease catalytic domain).

[0162] Competition between binder polypeptides can also be determined. For example, a binder polypeptide can compete with an antibody (e.g., an 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. A binder polypeptide can compete with, for example, NORI-003 scFv. A binder polypeptide can compete with NORI-006 scFv. A binder polypeptide can compete with NORI-011 scFv. A binder polypeptide can compete with NORI-008 scFv.

[0163] Competition between binder polypeptides indicates that they have epitopes within the same region of MTP-2, eg, they can both bind to the same domain with overlapping binding footprints.

[0164] IC50 can be calculated in a competitive assay as an indicator of the ability of the binder polypeptide to inhibit the binding of a reference molecule (e.g., aprotinin or NORI antibody) to MTP-2.The binder polypeptide may have an IC50 of less than 100nM in such an assay.In some cases, the IC50 is less than 50nM, for example, less than 20nM.

[0165] For example, the IC50 for competition with aprotinin can be determined in an HTRF competition assay using directly labeled aprotinin (e.g., aprotinin-647) at a concentration of 5 nM, a binder polypeptide at a concentration of 0.3 nM, and either human MTP-2 antigen at a concentration of 10 nM or mouse MTP-2 antigen at a concentration of 60 nM. The secondary antibody (e.g., AD0207) can be used at a 1:1000 dilution. See Example 8, where aprotinin was directly labeled using the 647 rapid labeling kit (Innova Biosciences-362-0010) according to the manufacturer's instructions. To determine the IC50 for competition of a binder polypeptide with a reference NORI antibody, the reference antibody can be directly labeled (e.g., with 647 labeling) as described above and using the same protocol, with the reference antibody serving as a substitute for aprotinin.

[0166] Materials and methods for HTRF competition assay Antibodies were titrated at 4x concentrations in HTRF buffer (DPBS (Gibco-14190144) with 0.1% BSA (Sigma-A7906) and 0.53M potassium fluoride (Sigma-60240-250G)) at a starting concentration of 200 nM. 5 μL / w of antibody was added to a 384-well white plate (Greiner-784904). Purified huMTP-2 and moMTP-2 proteins were diluted to 4x final concentrations in HTRF buffer (huMTP-2 = 40 nM, and moMTP-2 = 240 nM) and plated at 5 μL / w. The moIgG1-based anti-MTP-2 mAb (NORI-037) was then diluted to a 4x final concentration of 1.2 nM with DELFIA Eu-N1 rabbit anti-mouse IgG antibody (AD0207) at a 1:1000 dilution in HTRF buffer. Finally, 647-labeled aprotinin (Sigma-A3428) was diluted to a 4x final concentration of 20 nM in HTRF buffer and plated at 5 μL / w. The plate was incubated at RT in the dark for at least 3 hours. The plate was read at 1, 2, and 3 hours using an HTRF 100-flash protocol on an EnVision plate reader (Ex: 340 nm, Em1: 620 nm, Em2: 665 nm).

[0167] Binding affinity The binder polypeptide has an affinity (K) for human MTP-2 of less than 100 nM, less than 50 nM, less than 25 nM, or less than 10 nM. D The binder polypeptide may have an affinity (Kd) for mouse MTP-2 of less than 100 nM, less than 50 nM, less than 25 nM, or less than 10 nM. The binder polypeptide may have an affinity (Kd) for rat MTP-2 of less than 100 nM, less than 50 nM, less than 25 nM, or less than 10 nM. D The binder polypeptide may have an affinity (Kd) for cynomolgus MTP-2 of less than 100 nM, less than 50 nM, less than 25 nM, or less than 10 nM.

[0168] In some embodiments, the K Dcan 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. D is optionally at least 0.001 nM, such as at least 0.005 nM.

[0169] The binder polypeptides may have affinities (K) within the ranges shown in Example 6, e.g., Table K or Table W. D The K of the binder polypeptide for binding MTP-2 can be D is the K of any of the NORI-001 to NORI-033 IgG or scFv (e.g., NORI-009 IgG). D is equal to or lower than K D is the K of aprotinin D is equal to or lower than

[0170] The affinity of a binder polypeptide for MTP-2 can be quantified in terms of the equilibrium dissociation constant, KD, which is the ratio of the association or on-rate (Ka) to the dissociation or off-rate (kd) of the binding interaction, Ka / Kd. KD, Ka, and Kd for antigen binding can be measured using surface plasmon resonance (SPR). Exemplary SPR procedures and conditions are provided in Example 6.

[0171] Briefly, SPR can be performed at 25°C by capturing binder polypeptides onto the chip at a concentration of 1 μg / ml for 60 seconds at 10 μl / min (approximately 35-50 RU captured), injecting MTP-2 (analyte) at 30 μl / min for 120 seconds (association time), and monitoring dissociation for 600 seconds. Analytes can be injected at concentrations of 100, 25, 6.25, 1.56, and 0 nM. Sensorgrams of binder polypeptides are generated, and the data are fitted to a 1:1 interaction model (e.g., RI=0 using Biacore evaluation software with globally fitted Rmax, k, kd).

[0172] Affinity quantification can be performed using SPR with antigen-binding polypeptide arms in monovalent form, such as antibody Fab or Fv containing an antigen-binding site, or heterodimeric immunoglobulins (e.g., IgG) with a single antigen-binding arm for the antigen of interest. Alternatively, it may be convenient to determine affinity to antigen-binding polypeptide arms in bivalent form, such as IgG containing homodimeric antigen-binding arms. SPR can involve coating dimers of antigen-binding polypeptide arms (directly or indirectly) on a biosensor chip, exposing the antigen-binding polypeptide arms to antigen in buffer solutions of various concentrations, detecting binding, and calculating the equilibrium dissociation constant, KD, of the binding interaction. SPR can be performed at 25°C. A suitable buffer solution is 150 mM NaCl, 0.05% surfactant (e.g., P20), and 3 mM EDTA, pH 7.6. HBS-P 1x (HEPES 10 mM, pH 7.4, NaCl 2.5 mM) containing CaCl2. 150 mM, EDTA 3 mM, 0.05% polysorbate 20, pH 7.6) is an exemplary buffer. Binding data can be fitted to a 1:1 model using standard algorithms, which may be specific to the instrument used. A variety of SPR instruments are known, such as Biacore™, ProteOn XPR36™ (Bio-Rad®), and KinExA® (Sapidyne Instruments, Inc.).

[0173] As described elsewhere herein, isolated and purified MTP-2 ECD is conveniently used in assays and is a suitable analyte for SPR.

[0174] Cross-reactivity Regulatory agencies may require that candidate therapeutic molecules demonstrate therapeutic efficacy in laboratory animals before proceeding to human clinical trials. Examples of mouse models of beta-thalassemia and evaluation of binder polypeptides in wild-type mice are described herein. To enable testing of binder polypeptides in such animal models, it is desirable to make the binder cross-reactive with corresponding antigens from one or more non-human mammals. Thus, the binder should be capable of cross-reacting with both non-human MTP-2 and human MTP-2. P-2 may also bind.

[0175] One way to quantify the degree of interspecies cross-reactivity of an antigen-binding molecule (or, more precisely, its antigen-binding site) is to measure the fold difference in its affinity to an antigen of one species compared to that of another, for example, the fold difference in affinity to a human antigen and a mouse antigen. Affinity can be quantified as KD, which refers to the equilibrium dissociation constant of the binding of an antigen to an antigen-binding molecule. KD can be determined by SPR, as described elsewhere herein.

[0176] Species cross-reactive binding molecules may have a percent difference in affinity for binding to human and non-human antigens of 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. Stated another way, the KD for binding of the extracellular domain of a human antigen may be within 30-fold, 25-fold, 20-fold, 15-fold, 10-fold, or 5-fold of the KD for binding of the extracellular domain of a non-human antigen.

[0177] Preferably, the binding affinity of the human and non-human antigens is within a range of 10-fold or less, more preferably within 5-fold or 2-fold. For example, the KD of binding of the non-human MTP-2 is up to 10-fold (preferably up to 5-fold or up to 2-fold) greater or up to 10-fold less (preferably up to 5-fold or up to 2-fold less) than the Kd of binding of human MTP-2, as determined by surface plasmon resonance.

[0178] A binding molecule can also be considered cross-species reactive if the KD for binding to both antigens meets a threshold, e.g., if the KD for binding to the human antigen and the KD for binding to the non-human antigen are both 10 mM or less, preferably 5 mM or less, and 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.

[0179] Cross-reactivity can also be identified by the ability of a binder polypeptide to recognize multiple species of MTP-2 expressed on cells, for example, using FACS. HTRF can also be used to determine binding and cross-reactivity with multiple species of MTP-2.

[0180] Binder polypeptides may have some ability to block enzymatic activity via fluorescent readout using multiple species of MTP-2 substrates (e.g., human and one or more or all of mouse, rat, and cynomolgus monkey MTP-2). Binder polypeptides may exhibit dose-dependent inhibition of MTP-2 catalytic activity in assays described herein using human and non-human (e.g., mouse, rat, or cynomolgus monkey) MTP-2.

[0181] Although interspecies cross-reactivity for binding antigens of different species can be advantageous, selectivity of the binder for MTP-2 is still necessary to avoid undesirable side effects. Therefore, MTP-2 is preferably the only antigen in the body to which the antigen-binding site of the binder polypeptide binds. Despite this, the binder polypeptide may optionally be engineered to contain additional binding sites, and antibodies containing antibody constant regions may optionally bind to, for example, one or more Fc receptors.

[0182] Optionally, the binder polypeptide does not bind to MTP-1 (e.g., human MTP-1). Optionally, the binder polypeptide does not bind to MTP-3 (e.g., human MTP-3). Optionally, the binder polypeptide does not bind to other members of the type II transmembrane serine protease family.

[0183] Inhibition of MTP-2 MTP-2 is primarily expressed in hepatocytes and plays a major role in iron metabolism by regulating hepcidin expression from hepatocytes. It has now been shown that hepcidin expression, a key regulator of iron homeostasis, is controlled by bone morphogenetic protein (BMP) growth factors, which bind to type I and type II BMP receptors in hepatocytes and induce the BMP / sons of mothers against decapentaplegic (SMAD) signaling pathway. Phosphorylation of the Smad1, 5, and 8 / Smad4 complex downstream of the BMP receptor increases the expression of the HAMP gene encoding hepcidin, leading to increased hepcidin secretion. Hepcidin binds to the iron transporter ferroportin present in duodenal cells, macrophages, and hepatocytes, inducing its internalization and degradation, thereby reducing the amount of iron entering the blood and thereby reducing blood iron levels. MTP-2 is thought to negatively regulate hepcidin expression by selectively cleaving members of the BMPR complex on the hepatocyte surface, thus silencing BMP / SMAD signaling. One proposed enzymatic target of MTP-2 is hemojuvelin (HJV), a co-receptor of the BMPR complex required for maximal BMP / SMAD signaling. However, although studies have shown that MTP-2 can cleave HJV and generate specific cleavage products, MTP-2-masked mice exhibited a reciprocal reduction in the expression of membrane-bound HJV, whereas TMPRSS6 expression was significantly reduced. KO mice show increased levels of cleaved HJV. More recently, studies have shown that MTP-2 most likely cleaves multiple members of the BMPR complex to inhibit hepcidin expression.

[0184] Tissue-wide scans of TMPRSS6 mRNA expression also reveal low levels of MTP-2 expression in the testis, but the role of MTP-2 there is largely unknown. Because of the local expression of MTP-2, both TMPRSS6 KO mice and humans with loss-of-function MTP-2 mutations exhibit a phenotype with greatly elevated iron levels and no additional side effects, suggesting that its additional role for iron regulation is limited and therefore less of a concern for anti-MTP-2 therapy.

[0185] The present invention therefore proposes to inhibit MTP-2, thereby preventing or reducing the cleavage of its downstream substrates, thereby reducing MTP-2 inhibition of hepcidin expression.

[0186] Inhibition of MTP-2 refers to inhibition of the enzymatic activity of MTP-2. MTP-2 is a serine protease, and inhibitors can inhibit the catalysis of serine protease cleavage of its substrate by mature, active MTP-2 and / or inhibit the autoactivation of MTP-2 zymogen by catalysis of serine protease cleavage.

[0187] In various embodiments, the binder polypeptide can bind to the serine protease catalytic domain of MTP-2. The serine protease catalytic domain contains the enzymatic active site of MTP-2. Inhibition can be the result of steric hindrance of the enzyme-substrate interaction caused by the binder polypeptide binding to MTP-2 and partially or completely masking the enzymatic active site, thereby reducing substrate binding. Alternatively, or in addition, inhibition can be the result of the binder polypeptide inducing an inactivating conformational change in the serine protease catalytic domain or biasing the serine protease catalytic domain toward an inactive conformation, thereby reducing its enzymatic activity. Regardless of the molecular mechanism of inhibition, the ability of the binder polypeptide to inhibit MTP-2 serine protease activity can be functionally determined in an enzyme assay.

[0188] This specification describes in vitro assays for inhibiting serine protease cleavage of MTP-2 substrates to produce detectable products. These include an enzyme assay using purified MTP-2 ECD and a fluorescent substrate, and an enzyme assay using cell surface-expressed MTP-2 and a fluorescent substrate. Each of these assays determines the inhibition of MTP-2 cleavage of its substrate. The fluorescent substrate Boc-Gln-Gly-Arg-AMC can be used at a final concentration of, for example, 50 μM. Binder polypeptides that are inhibitors of MTP-2 enzyme activity are identified by dose-dependent inhibition of MTP-2 serine protease activity in such enzyme assays.

[0189] In one embodiment, the binder polypeptide has an IC50 of less than 100 nM in an enzyme assay against the extracellular domain of human MTP-2, with an activity rate of 0.075 U / μl in the presence of 50 μM Boc-Gln-Gly-Arg-AMC fluorescent substrate. The IC50 may 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 may optionally be 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.

[0190] In one embodiment, the binder 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 an activity rate of 0.075 U / μl in the presence of 50 μM Boc-Gln-Gly-Arg-AMC fluorescent substrate. The IC50 may 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.

[0191] As noted above, preferably, the binder polypeptides are species cross-reactive, and thus inhibitory to MTP-2 of more than one species, e.g., human and non-human (e.g., mouse, rat, or cynomolgus monkey) MTP-2. Parameters for quantifying cross-reactivity in assays are discussed elsewhere herein.

[0192] In one embodiment, the binder polypeptide exhibits dose-dependent inhibition of MTP-2 serine protease activity in an enzyme assay using human MTP-2 expressed on the surface of HEK293 cells and a fluorescent MTP-2 substrate at a final concentration of 50 μM. The binder polypeptide may have an IC50 of less than 100 nM in the assay. The IC50 may 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.

[0193] Inhibition of the enzymatic activity of MTP-2 can also be detected in hepatocytes. For example, binder polypeptides that inhibit MTP-2 can increase hepcidin expression in hepatoma cell lines (with or without bmp stimulation), which can be measured as an increase in hamp mRNA compared to controls.

[0194] Similar readouts can be obtained from in vivo assays. In mice administered with binder polypeptides, inhibition of the enzymatic activity of MTP-2 can result in increased hamp mRNA, reduced serum iron, and reduced transferrin saturation (TSAT). To measure these effects, binder polypeptides are administered to wild-type mice (e.g., at 10 mg / kg). Binder polypeptides increase hamp mRNA levels in hepatocytes within 24 hours after administration. The binder polypeptides may increase hamp mRNA by at least twofold, and this increase may persist for 3 days, preferably 21 days, after administration (e.g., after a single 10 mg / kg intraperitoneal dose). The binder polypeptides may reduce serum iron levels in mice. Again, this may be detectable within 24 hours, and preferably persist for 3 days, preferably 21 days, after administration. Exemplary experiments and protocols for measuring hamp mRNA and quantifying serum iron and TSAT are provided in the Examples.

[0195] Binder polypeptides A binder polypeptide according to the present invention is a polypeptide molecule that has the ability to bind to and inhibit MTP-2.

[0196] Many classes of binder polypeptides are known in the art, including traditional IgG antibodies and other binding proteins based on immunoglobulin domains (see Binz, Amstutz, and Pluckthun, Nature Biotechnology 23(10):1257, 2005). Molecules that do not bind immunoglobulins are also known, and binding loops can be engineered into other polypeptide scaffolds, such as fibronectin.

[0197] Preferably, the binder polypeptide of the present invention comprises an immunoglobulin domain in which the binding site for MTP-2 is formed by a loop region of the immunoglobulin domain.A preferred embodiment of the binder polypeptide is an antibody.

[0198] Antibodies according to the present invention are immunoglobulins or molecules containing immunoglobulin domains, either natural or partially or wholly synthetically produced. Antibodies can be IgG, IgM, IgA, IgD, or IgE molecules, or antigen-specific antibody fragments thereof (including, but not limited to, Fab, F(ab'), Fv, disulfide-linked Fv, scFv, single-domain antibodies, closed-conformation multispecific antibodies, disulfide-linked scFv, and diabodies), derived from any species that naturally produces antibodies or produced by recombinant DNA technology; isolated from serum, B cells, hybridomas, transfectomas, yeast, or bacteria. Antibodies can be humanized using conventional techniques. The term antibody encompasses any polypeptide or protein that contains an antibody antigen-binding site. The antigen-binding site (paratope) is the portion of an antibody that binds to and is complementary to the epitope (MTP-2) of its target antigen.

[0199] The term "epitope" refers to the region of an antigen to which an antibody binds. Epitopes are defined as either structural or functional. Functional epitopes are generally a subset of structural epitopes and contain residues directly involved in the affinity of the interaction. Epitopes can also be conformational, i.e., composed of nonlinear amino acids. In certain embodiments, epitopes can include antigenic determinants that are chemically active molecular surface groups such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, can have specific three-dimensional structural characteristics and / or specific charge characteristics.

[0200] An antigen-binding site is a polypeptide or domain that contains one or more CDRs of an antibody and can bind to an antigen. For example, the polypeptide contains CDR3 (e.g., HCDR3). For example, the polypeptide contains CDR1 and 2 (e.g., HCDR1 and 2) or CDR1-3 (e.g., HCDR1-3) of the variable domain of an antibody.

[0201] The antigen-binding site of an antibody is provided by one or more antibody variable domains. In one example, the binding site of an antibody is provided by a single variable domain, such as a heavy chain variable domain (VH domain) or a light chain variable domain (VL domain). In another example, A binding site comprises a VH / VL pair, or two or more such pairs. Thus, the antigen-binding site of an antibody may comprise a VH and a VL.

[0202] An antibody can be a whole immunoglobulin, including the constant region, or it can be an antibody fragment. An antibody fragment is a portion of an intact antibody, for example, including the antigen-binding and / or variable region of the intact antibody. Examples of antibody fragments include: (i) Fab fragment, i.e., a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bond at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of a VH or VL domain (Ward et al. (1989) Nature 341:544-546, incorporated herein by reference in its entirety); and (vi) an isolated complementarity-determining region (CDR) that retains specific antigen-binding functionality; Examples include:

[0203] A further example of an antibody is the H2 antibody, which comprises a dimer of heavy chains (5'-VH-(optional hinge)-CH2-CH3-3') and lacks light chains.

[0204] Single-chain antibodies (e.g., scFv) are commonly used fragments. Multispecific antibodies are formed from antibody fragments. The antibodies of the present invention may utilize any such format as desired.

[0205] Optionally, the binder polypeptide, or its antibody immunoglobulin domain, can be fused or conjugated to additional polypeptide sequences and / or to labels, tags, toxins, or other molecules. The binder polypeptide can be fused or conjugated to one or more different antigen-binding regions to provide a molecule that can bind to a second antigen in addition to MTP-2. For example, the antibody of the present invention can be a multispecific antibody, e.g., a bispecific antibody, comprising (i) an antibody antigen-binding site for MTP-2 and (ii) an additional antigen-binding site that recognizes another antigen (optionally an antibody antigen-binding site described herein).

[0206] An antibody typically comprises an antibody VH and / or VL domain. Isolated antibody VH and VL domains are also part of the present invention. An antibody variable domain is a portion of an antibody's light and heavy chains, comprising the amino acid sequences of complementarity-determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) and framework regions (FRs). Thus, each VH and VL domain has CDRs and FRs. A VH domain contains a set of HCDRs, and a VL domain contains a set of LCDRs. VH refers to the heavy chain variable domain. VL refers to the light chain variable domain. Each VH and VL typically consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. According to the method used in the present invention, the amino acid positions assigned to CDRs and FRs are defined according to Kabat (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991)) or according to the IMGT nomenclature.

[0207] The antibody may comprise an antibody VH domain comprising CDR1, CDR2, and CDR3 of the VH, and a framework. The antibody may alternatively, or in addition, comprise CDR1, CDR2, and CDR3 of the VL. The antibody VH and VL domains may include antibody VL domains comprising CDR1, CDR2, and CDR3, and frameworks. Examples of VH and VL domains and CDRs of antibodies according to the present invention are listed in Table S. All VH and VL sequences, CDR sequences, sets of CDRs, and sets of HCDRs and sets of LCDRs disclosed herein are aspects and embodiments of the present invention. As described herein, a "set of CDRs" includes CDR1, CDR2, and CDR3. Thus, a set of HCDRs refers to HCDR1, HCDR2, and HCDR3, and a set of LCDRs refers to LCDR1, LCDR2, and LCDR3. Unless otherwise specified, a "set of CDRs" includes HCDRs and LCDRs.

[0208] As described in more detail in the Examples, the inventors have identified 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- Antibodies of particular interest, designated 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 through NORI-033"), have been isolated and characterized.

[0209] In various embodiments of the invention, unless the context dictates otherwise, the antibody may be selected from any of these antibodies or from a subset of NORI-003, NORI-006, NORI-011, or NORI-008.

[0210] The present invention encompasses anti-MTP-2 antibodies having the VH and / or VL domain sequences of all antibodies shown in the accompanying sequence listing and / or figures, as well as antibodies comprising the HCDRs and / or LCDRs of these antibodies, and optionally the entire heavy chain and / or light chain amino acid sequences of any anti-MTP-2 antibody disclosed herein.

[0211] CDR sequences are defined by IMGT or by other methods such as Kabat. Unless otherwise specified, references to residues in the variable domain or to CDR or framework regions refer to the IMGT definition.

[0212] When the antibody VH or VL domain contains one or more residues in the framework regions that differ from the germline gene segment from which it was derived by recombination, the non-germline residues can be retained or mutated to a different residue, e.g., a non-germline residue can be reverted to a germline residue. The corresponding germline gene segment is identified as the gene segment to which the sequence of the variable domain most closely aligns, and the germline gene segments corresponding to each of the VH and VL domains of NORI-001 through NORI-033 are set forth herein in Table G.

[0213] Antibodies according to the invention may comprise one or more CDRs described herein, such as CDR3, and optionally may also comprise CDR1 and CDR2 to form a set of CDRs. The CDR or set of CDRs may be that of any of NORI-001 to NORI-033.

[0214] The present invention relates to the HCDR1, HCDR2, and / or HCDR3 of any of antibodies NORI-001 to NORI-033, and / or the L of any of these antibodies. Antibodies are provided that comprise a set of CDRs, such as CDR1, LCDR2, and / or LCDR3. The antibody may comprise a set of VH CDRs of one of these antibodies. Optionally, the antibody may also comprise a set of VL CDRs of one of these antibodies, where the VL CDRs are VH CDRs. The CDRs may be from the same or different antibodies.

[0215] Also provided by the invention are VH domains comprising the disclosed sets of HCDRs, and / or VL domains comprising the disclosed sets of LCDRs.

[0216] As discussed further below, although either the VH or VL domain alone can be used to bind to an antigen, typically the VH domain pairs with the VL domain to form the antigen-binding site of an antibody. The VH domain of NORI-003 can pair with the VL domain of NORI-003, thus forming an antibody antigen-binding site comprising both the VH and VL domains of NORI-003. 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. Promiscuous light chain pairing is well known in the art. Again, similar embodiments are provided by the present invention for the other VH and VL domains disclosed herein.

[0217] Thus, the VH of any of the antibodies NORI-001 to NORI-033 can pair with the VL of any of the antibodies NORI-001 to NORI-033.

[0218] An antibody can contain one or more CDRs, e.g., a set of CDRs, within an antibody framework. The framework regions can be of human germline gene segment sequences. Thus, an antibody can be a human antibody having a VH domain containing a set of HCDRs in a human germline framework. Typically, an antibody also has a VL domain containing a set of LCDRs, e.g., 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 a nucleic acid sequence from which that portion of an antibody is derived. For example, a VH gene segment is an oligonucleotide containing a nucleic acid sequence corresponding to a portion of FR1 through CDR3 of a polypeptide VH domain. Human V, D, and J gene segments recombine to generate a VH domain, and human V and J segments recombine to generate a VL domain. A D domain or D region refers to the diversity domain or region of an antibody chain. A J domain or J region refers to the joining domain or region of an antibody chain. Although somatic hypermutation can result in antibody VH or VL domains with framework regions that do not exactly match or align with the corresponding gene segments, sequence alignment can be used to identify the closest gene segments and, therefore, from which particular combination of gene segments a particular VH or VL domain is derived. When aligning an antibody sequence with a gene segment, the amino acid sequence of the antibody is aligned with the amino acid sequence encoded by the gene segment, or the nucleotide sequence of the antibody is aligned directly with the nucleotide sequence of the gene segment.

[0219] The antibodies of the present invention can be human antibodies or chimeric antibodies comprising human variable regions and non-human (e.g., murine) constant regions, e.g., antibodies of the present invention have human variable regions and, optionally, also have human constant regions.

[0220] Thus, an antibody optionally comprises a constant region or a portion thereof, such as the constant region or a portion thereof of a human antibody. For example, a VL domain may be attached at its C-terminus to an antibody light chain kappa or lambda constant domain. Similarly, an antibody VH domain may be attached at its C-terminus to an antibody light chain kappa or lambda constant domain. At its terminus, it may be attached all or part of an immunoglobulin heavy chain constant region (e.g., CH1 domain or Fc region) from any antibody isotype, e.g., IgG, IgA, IgE, and IgM, and any of the isotype subclasses, e.g., IgG1 or IgG4.

[0221] Examples of human heavy chain constant regions are shown in Table S.

[0222] The constant regions of the antibodies of the present invention can alternatively be non-human constant regions. For example, when antibodies are produced in transgenic animals (examples of which are described elsewhere herein), chimeric antibodies containing human variable regions and non-human (host animal) constant regions are produced. Some transgenic animals produce fully human antibodies. Others have been engineered to produce antibodies containing chimeric heavy chains and fully human light chains. When antibodies contain one or more non-human constant regions, replacing them with human constant regions reduces their immunogenicity, and such replacement may be performed to produce antibodies more suitable for administration to humans as therapeutic compositions.

[0223] Digestion of antibodies with the enzyme papain produces two identical antigen-binding fragments, also known as "Fab" fragments, and an "Fc" fragment, which lacks antigen-binding activity but retains crystallization ability. As used herein, "Fab" refers to an antibody fragment containing one constant domain and one variable domain from each of the heavy and light chains. The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. "Fc fragment" refers to the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by the sequence in the Fc region, which is also recognized by Fc receptors (FcRs) found on certain types of cells. Digestion of antibodies with the enzyme pepsin produces an F(ab')2 fragment, in which the two arms of the antibody molecule remain linked and contain two antigen-binding sites. The F(ab')2 fragment has the ability to cross-link antigens.

[0224] As used herein, "Fv" refers to the minimum fragment of an antibody that retains both the antigen recognition and binding sites. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain tightly bound by non-covalent or covalent bonds. In this conformation, 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 containing only three antigen-specific CDRs) has the ability to recognize and bind to antigen, albeit with lower affinity than the entire binding site.

[0225] The antibodies disclosed herein are modified to increase or decrease serum half-life. In one embodiment, one or more of the following mutations are introduced to increase the biological half-life of the antibody: T252L, T254S, or T256F. As described in U.S. Patent Nos. 5,869,046 and 6,121,022, the modifications described therein are incorporated herein by reference, the biological half-life can also be increased by modifying the CH1 or CL region of the heavy chain constant region to contain a salvage receptor binding epitope obtained from two loops of the CH2 domain of the Fc region of IgG. In another embodiment, the Fc hinge region of the antibody or antigen-binding fragment of the present invention 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 compared to the SpA binding of the native Fc-hinge domain. Other methods of increasing serum half-life are known to those skilled 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, e.g., 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 (XL-Protein GmbH), which forms an uncharged random coil structure with a large hydrodynamic volume). In another embodiment, the antibody or fragment is XTENylated® / rPEGylated (i.e., genetic fusion of an imprecise repeat peptide sequence (Amunix, Versartis) to a therapeutic peptide). In another embodiment, the antibody or fragment is ELPylated (i.e., genetic fusion to an ELP repeat sequence (PhaseBio)). These various half-life-extending fusions are described in more detail in Strohl, BioDrugs (2015) 29:215-239, e.g., the fusions in Tables 2 and 6, which are incorporated herein by reference.

[0226] antibody constant region As mentioned above, antibodies are provided in various isotypes and with different constant regions. The Fc region of an antibody is recognized by Fc receptors and determines the antibody's ability to mediate cellular effector functions, including antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP). These cellular effector functions involve recruiting Fc receptor-bearing cells to the site of target cells and killing the cells bound to the antibody.

[0227] In the context of the present invention, it is desirable to avoid cellular effector functions such as ADCC, ADCP, and / or CDC. Thus, antibodies according to the present invention may lack Fc effector function; for example, antibodies according to the present invention may have an Fc region that does not mediate ADCC, ADCP, and / or CDC, or they may lack an Fc region or the entire antibody constant region. The antibody may have an effector-null constant region.

[0228] An antibody can have a heavy chain constant region that binds to one or more types of Fc receptor but does not induce cellular effector function, i.e., does not mediate ADCC, CDC, or ADCP activity. Such a constant region may not be able to bind to the particular Fc receptor responsible for triggering ADCC, CDC, or ADCP activity.

[0229] The antibody may have a heavy chain constant region that does not bind to Fcγ receptors, for example, the constant region may contain an "E" mutation, such as a Leu235Glu mutation (i.e., the wild-type leucine residue is mutated to a glutamic acid residue), referred to as IgG4-E. Another optional mutation in the heavy chain constant region is Ser228Pro (a "P" mutation), which increases stability by reducing Fab arm exchange. The heavy chain constant region may be an IgG4 containing both the Leu235Glu mutation and the Ser228Pro mutation. This "IgG4-PE" heavy chain constant region is effector-null. An alternative effector-null human constant region is a disabled IgG1.

[0230] IgG4PE is a preferred antibody isotype of the present invention. The binder polypeptide can be an IgG4PE antibody comprising the sequence of the IgG4PE constant region shown in Table S.

[0231] Antibody constant regions are engineered to increase their in vivo half-life. Examples include the "YTE" mutation and other half-life extending mutations (Dall'Acqua, Kiener, and Wu, JBC 281(33):23514-23524, 2006, and WO02 / 060919, which are incorporated herein by reference). The triple mutation YTE is a substitution of three amino acids in the CH2 domain of IgG, and these The 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. As described in the referenced publications, the YTE modification increases the half-life of the antibody compared to the half-life of a corresponding antibody having a wild-type human CH2 domain. To increase in vivo shelf life, the antibodies of the invention may comprise an antibody constant region (e.g., an IgG constant region, e.g., an IgG CH2 domain) with one or more mutations that increase the half-life of the antibody compared to a corresponding wild-type human constant region (e.g., an IgG, e.g., an IgG CH2 domain). Half-life can be determined by standard methods, such as those described in WO 02 / 060919.

[0232] The appropriate antibody constant region can be selected according to the genotype of the patient to be treated. For example, as described in US20160319017, a method for increasing erythropoiesis in a human patient includes: rs855791, rs2543519, rs2235324, and rs1421312 administering a binder (e.g., an antibody) that binds to human MTP-2 encoded by a TMPRSS6 nucleotide sequence containing a SNP selected from the group consisting of: The binder is (a) a heavy chain constant region of human gamma-4 comprising Leu at position 189 of SEQ ID NO: 73 or Arg at position 289 of SEQ ID NO: 73 of US20160319017; and (b) a heavy chain constant region of human gamma-1, 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; wherein: (i) the human subject comprises a TMPRSS6 nucleotide sequence comprising the selected SNP; and (ii) the human patient comprises a constant region gene segment encoding said selected constant region; or the human patient expresses an antibody comprising said selected constant region.

[0233] The antibodies described in US20160319017 may comprise the constant regions described in said publication and / or may be used to treat patients containing the TMPRSS6 nucleotide sequence polymorphisms described in said publication.

[0234] Further exemplary constant regions are shown in Table S.

[0235] Production and modification of binder polypeptides Methods for identifying and producing binder polypeptides, including antibodies, are well known in the art.

[0236] For example, antibodies can be generated using experimental animals, e.g., mice, including transgenic mice (e.g., Kymouse™, Velocimouse®, Omnimouse®, Xenomouse®, HuMab Mouse®, or MeMo Mouse®), rats (e.g., Omnirat®), camelids, sharks, rabbits, chickens, or other non-human animals, that have been immunized with MTP-2 or a fragment thereof (e.g., recombinant MTP-2 ECD) or its encoding nucleic acid, followed by humanization of the constant and / or variable regions, optionally to produce human or humanized antibodies. In one example, display technologies such as yeast, phage, or ribosome display are used, as will be apparent to those skilled in the art. For example, standard affinity maturation using display technologies can be performed using transgenic animals, phage display, or other non-human animals. Further steps are performed after isolating antibodies derived from a single or other library. Representative examples of suitable techniques are described in US20120093818 (Amgen, Inc.), the entire contents of which are incorporated herein by reference, e.g., in the methods set forth in paragraphs

[0309] to

[0346] .

[0237] There are many reasons why it may be desirable to create variants of a binder, including optimizing the polypeptide sequence for large-scale manufacturing, facilitating purification, enhancing stability, or improving suitability for inclusion in a desired pharmaceutical formulation. For example, protein engineering work can be performed at one or more target residues in an antibody sequence, such as substituting an amino acid with an alternative amino acid (potentially generating variants containing all naturally occurring amino acids at this position, with the possible exception of Cys and Met), and monitoring the effects on function and expression to determine the best substitution. Substituting a residue with Cys or Met or introducing these residues into the sequence may not be desirable because doing so can cause manufacturing problems, for example, due to the formation of new intramolecular or intermolecular cysteine-cysteine ​​bonds. Once a lead candidate has been selected and optimized for manufacturing and clinical development, it will generally be desirable to minimize its antigen-binding properties, or at least retain the affinity and potency of the parent molecule. However, variants can also be generated to adjust important antibody characteristics, such as affinity, cross-reactivity, or neutralization potency.

[0238] The antibody can comprise a set of H and / or L CDRs of any of the disclosed antibodies, including 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 a 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 may comprise a set of HCDRs, LCDRs, or a set of six (H and L) CDRs set forth for any NORI antibody herein, or may comprise that set of CDRs containing one or two conservative substitutions.

[0239] One or more amino acid mutations are optionally introduced into 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 are reverted to the germline. Human germline gene segment sequences corresponding to the VH and VL domains of exemplary anti-MTP-2 antibodies are shown in Table G.

[0240] The antibody may comprise a VH domain having at least 60, 70, 80, 85, 90, 95, 98, or 99% amino acid sequence identity with the VH domain of any of the antibodies set out in the accompanying sequence listing, and / or may comprise a VL domain having at least 60, 70, 80, 85, 90, 95, 98, or 99% amino acid sequence identity with the VL domain of any of those antibodies. Algorithms that can be used to calculate the percent identity of two amino acid sequences include, for example, BLAST, FASTA, or the Smith-Waterman algorithm, for example, using default parameters. Particular variants may contain one or more amino acid sequence alterations (addition, deletion, substitution, and / or insertion of amino acid residues).

[0241] The changes may affect one or more framework regions and / or one or more of the Modifications are made to the above CDRs. Mutations are optionally provided by CDR mutagenesis. The modifications do not usually result in loss of function, so that antibodies containing such modified amino acid sequences can retain the ability to bind to MTP-2. The antibodies can retain the same quantitative binding ability as antibodies that do not undergo modification, for example, as measured by the assays described herein. Antibodies containing such modified amino acid sequences can have improved abilities to bind to and / or inhibit MTP-2.

[0242] Alterations may include replacing one or more amino acid residues with non-naturally occurring or non-standard amino acids, modifying one or more amino acid residues to 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 invention are described elsewhere herein. Naturally occurring amino acids include the 20 "standard" L-amino acids, identified by the standard one-letter abbreviations G, A, V, L, I, M, P, F, W, S, T, N, Q, Y, C, K, R, H, D, and E. Non-standard amino acids include any other residue that can be incorporated into a polypeptide backbone or result from the modification of an existing amino acid residue. Non-standard amino acids may be naturally occurring or non-naturally occurring.

[0243] The term "variant," as used herein, refers to a peptide or nucleic acid that differs from a parent polypeptide or nucleic acid by the deletion, substitution, or addition of one or more amino acids or nucleic acids, but nonetheless retains one or more specific functions or biological activities of the parent molecule. Amino acid substitutions include alterations in which an amino acid is replaced with a different naturally occurring amino acid residue. Such substitutions are classified as "conservative," in which an amino acid residue contained in a polypeptide is replaced with another naturally occurring amino acid of similar characteristics, either in terms of 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 an amino acid residue present in the peptide is replaced with an amino acid having different properties, for example, a naturally occurring amino acid from a different group (e.g., replacing a charged or hydrophobic amino acid with alanine), or alternatively, a naturally occurring amino acid is replaced with an unconventional amino acid. In some embodiments, the amino acid substitution is conservative. Also encompassed within the term variant, when used with respect to a polynucleotide or polypeptide, is a polynucleotide or polypeptide that may differ in primary, secondary, or tertiary structure compared to a reference polynucleotide or polypeptide, respectively (e.g., compared to a wild-type polynucleotide or polypeptide).

[0244] In some embodiments, "synthetic," "recombinant," or "chemically modified" polynucleotide or polypeptide variants isolated or produced using methods well known in the art can be used. "Modified variants" may 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 a polypeptide encoded by a reference sequence. Some embodiments use insertional, deletional, or substitutional variants with amino acid substitutions that do not normally occur in the peptide sequence on which the variant is based, including insertions and substitutions of amino acids and other molecules that do not normally occur in human proteins, for example, but not limited to, the insertion of ornithine, which does not normally occur in human proteins. The term "conservative substitution," when referring to a polypeptide, refers to a change in the amino acid composition of a polypeptide that does not substantially alter the activity of the polypeptide. For example, a conservative substitution refers to the substitution of an amino acid residue for a different amino acid residue with 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, replacing aspartic acid with glutamic acid, or replacing threonine with serine. Conservative substitutions that provide functionally similar amino acids Tables are well known in the art. For example, the following six groups each contain amino acids that are conservative substitutions for 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, individual substitutions, deletions, or additions that alter, add, or delete a single amino acid or a small percentage of amino acids can also be considered "conservative substitutions" if the changes do not reduce the activity of the peptide. Insertions or deletions typically range from about 1 to 5 amino acids. The selection of conservative amino acids is based on the position of the amino acid to be substituted in the peptide, for example, whether the amino acid is on the exterior of the peptide and exposed to the solvent, or on the interior and not exposed to the solvent.

[0245] Amino acids to replace existing amino acids can be selected based on the position of the existing amino acid, including solvent exposure (i.e., whether the amino acid is solvent exposed or located on the exterior of the peptide or polypeptide, compared to an amino acid that is not solvent exposed and is located internally). Such conservative amino acid substitution selection can be performed, for example, as described 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, are well known in the art. Thus, suitable conservative amino acid substitutions can be selected for amino acids on the exterior of the protein or peptide (i.e., amino acids exposed to the solvent), for example, but not limited to, the following substitutions are used: substitution of Y with F, substitution of T with S or K, substitution of P with A, substitution of E with D or Q, substitution of N with D or G, substitution of R with K, substitution of G with N or A, substitution of T with S or K, substitution of D with N or E, substitution of I with L or V, substitution of F with Y, substitution of S with T or A, substitution of R with K, substitution of G with N or A, substitution of K with R, substitution of A with S, K or P.

[0246] In alternative embodiments, conservative amino acid substitutions that are suitable for amino acids in the interior of a protein or peptide can also be selected, for example, conservative substitutions that are suitable for amino acids that are in the interior of a 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: Y is substituted with F, T is substituted with A or S, I is substituted with L or V, W is substituted with Y, M is substituted with L, N is substituted with D, G is substituted with A, T is substituted with A or S, D is substituted with N, I is substituted with L or V, F is substituted with Y or L, S is substituted with A or T, and A is substituted with S, G, T, or V. In some embodiments, non-conservative amino acid substitutions are also encompassed within the term variant.

[0247] The present invention includes methods for making antibodies containing VH and / or VL domain variants of the antibody VH and / or VL domains set forth in Table S. Such antibodies include: (i) the parent antibody VH domain is the VH domain of any of NORI-001 to NORI-033, or a VH domain comprising the heavy chain complementarity-determining region of any of these antibodies; providing an antibody VH domain which is an amino acid sequence variant of a parent antibody VH domain by adding, deleting, substituting or inserting one or more amino acids into the amino acid sequence of the parent antibody VH domain; (ii) optionally combining the VH domain thus provided with a VL domain to provide a VH / VL combination; (iii) testing the VH domains or VH / VL domain combinations thus provided to identify antibodies having one or more desired characteristics; The method can be prepared by a method comprising:

[0248] The VH domain may be the VH domain of NORI-003. The VH domain may be the VH domain of NORI-006. The VH domain may be the VH domain of NORI-011. The VH domain may be the VH domain of NORI-008.

[0249] Desirable characteristics include binding to human and / or non-human MTP-2. Antibodies are identified that have comparable or higher affinity for human and / or mouse MTP-2 compared to the parent antibody. Other desirable 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. Identification of antibodies with desirable characteristics can include identification of antibodies with functional attributes described herein, such as their affinity, cross-reactivity, specificity, or neutralization potency, any of which can be determined by the assays described herein.

[0250] When a VL domain is included in the method, the VL domain may be the VL domain of any of NORI-001 to NORI-033, or a variant provided by adding, deleting, substituting, or inserting one or more amino acids into the amino acid sequence of the parent VL domain, where the parent VL domain is the VL domain of any of NORI-001 to NORI-033, or a VL domain comprising the light chain complementarity-determining region of any of these antibodies. The VL domain may be the VL domain of the same antibody as the VH domain. The VL domain may be the VL domain of NORI-003. The VL domain may be the VL domain of NORI-006. The VL domain may be the VL domain of NORI-011. The VL domain may be the VL domain of NORI-008.

[0251] Methods for generating variant antibodies may optionally include making copies of the antibody or VH / VL domain combination. The method may further include expressing the resulting antibody. Nucleotide sequences corresponding to the desired antibody VH and / or VL domains may optionally be produced in one or more expression vectors. Suitable methods for expression, including recombinant expression in a host cell, are described in detail herein.

[0252] Encoding nucleic acids and methods of production An isolated nucleic acid encoding an antibody according to the present invention is provided. The nucleic acid may be DNA and / or RNA. Genomic DNA, cDNA, mRNA or other RNA, or any combination thereof, of synthetic origin, can encode the antibody.

[0253] The present invention provides constructs in the form of plasmids, vectors, transcription or expression cassettes comprising at least one polynucleotide as described above.Exemplary nucleotide sequences are included in the sequence listing.Reference to the nucleotide sequences described herein includes DNA molecules with the specified sequence, and also includes RNA molecules with the specified sequence, in which U is substituted for T, unless the context otherwise requires.

[0254] The present invention also provides recombinant host cells containing one or more nucleic acids encoding the antibodies. Methods of producing the encoded antibodies may include expression from the nucleic acids, for example, by culturing recombinant host cells containing the nucleic acids. The antibodies may then be obtained, isolated and / or purified using any suitable technique, and then used as needed. Production methods may also include culturing the product in a composition containing at least one additional component, such as a pharmaceutically acceptable excipient. The method may include formulating the compound into a product.

[0255] Systems for cloning and expression of 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, and transgenic plants and animals.

[0256] The expression of antibodies and antibody fragments in prokaryotic cells is well established in the art. A common bacterial host is Escherichia coli. Expression in eukaryotic cells in culture 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.

[0257] The vector may contain appropriate control sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes, and other sequences, as needed. The nucleic acid encoding the antibody is introduced into a host cell. The nucleic acid of the present invention can be integrated into the genome (e.g., chromosome) of the host cell. Integration can be facilitated by including sequences that promote recombination with the genome, according to standard techniques. 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 or, for insect cells, baculovirus. Introduction of nucleic acids into host cells, particularly eukaryotic cells, can use virus- or plasmid-based systems. Plasmid systems can be maintained episomally or integrated into host cells or artificial chromosomes. Integration can be by either random or targeted integration of one or more copies at single or multiple loci. For bacterial cells, suitable techniques include calcium chloride transformation, electroporation, and transfection using bacteriophage. Following introduction, the nucleic acid is expressed, e.g., by culturing the host cells under conditions for expression of the gene, and the binder polypeptide, e.g., antibody, may then optionally be isolated or purified.

[0258] Formulation and Administration Binder polypeptides and their encoding nucleic acid molecules according to the present invention will typically be provided in isolated form. The VH and / or VL domains and nucleic acids are provided purified from their natural environment or the environment in which they are produced. Isolated binder polypeptides and isolated nucleic acids will be free or substantially free of materials with which they are naturally associated, e.g., other polypeptides or nucleic acids with which they are found in vivo, or the environment in which they are produced (if such production is by in vitro recombinant DNA technology) (e.g., cell culture). Optionally, an isolated binder polypeptide or nucleic acid (1) is free of at least some other proteins with which it is normally found; (2) is essentially free of other proteins from the same source, e.g., the same species; (3) is expressed by cells from a different species; (4) is separated from at least about 50 percent of the polynucleotides, lipids, carbohydrates, or other materials with which it is naturally associated; (5) is operably associated (by covalent or non-covalent interactions) with polypeptides with which it is not naturally associated; or (6) is not naturally occurring.

[0259] Binder polypeptides or their encoding nucleic acids may be formulated with a diluent or adjuvant, or even isolated for practical purposes, e.g., if they are to be used to coat microtiter plates for use in immunoassays. When used in therapy, they are mixed with a carrier, if desired, and with a pharmaceutically acceptable carrier or diluent. As described elsewhere in this invention, other active ingredients can also be included in the therapeutic product. The binder polypeptides can be glycosylated naturally in vivo or by heterologous eukaryotic systems such as CHO cells, or can be non-glycosylated (e.g., when produced by expression in prokaryotic cells). The present invention encompasses antibodies with modified glycosylation patterns.

[0260] Typically, an isolated product comprises at least about 5%, at least about 10%, at least about 25%, or at least about 50% of a given sample. The binder polypeptide may be substantially free of proteins or polypeptides or other contaminants found in its natural or production environment that would interfere with its therapeutic, diagnostic, prophylactic, research, or other uses.

[0261] The present invention provides therapeutic compositions comprising the binder polypeptides described herein. Therapeutic compositions comprising nucleic acids encoding such binder polypeptides are also provided. Encoding nucleic acids are described in more detail elsewhere in this specification, and include DNA and RNA, e.g., mRNA. In the therapeutic methods described herein, the use of nucleic acids encoding binder polypeptides and / or cells containing such nucleic acids can be used as an alternative (or in addition) to compositions comprising the binder polypeptides themselves. Cells containing nucleic acids encoding binder polypeptides, optionally with the nucleic acid stably integrated into the genome, are therefore pharmaceuticals for therapeutic use in patients. Nucleic acids encoding binder polypeptides are introduced into human cells derived from the intended patient and modified ex vivo. Administration of cells containing encoding nucleic acids to a patient provides a reservoir of cells capable of expressing the binder polypeptide and may result in longer-lasting therapeutic benefits compared to administration of isolated nucleic acids or isolated binder polypeptides. Nucleic acids can also be administered directly to a patient for gene therapy. Thus, nucleic acids encoding binder polypeptides are provided for use in gene therapy, which involves introducing the encoding nucleic acid into the cells of a patient in vivo, such that the nucleic acid is expressed in the patient's cells and provides a therapeutic effect. Exemplary therapeutic effects are disclosed herein, including increasing hamp mRNA, lowering serum iron, lowering TSAT, and treating diseases and conditions associated with iron overload.

[0262] The compositions may contain suitable carriers, excipients, and other agents that are incorporated into the formulation to improve mobility, delivery, tolerability, etc. Many suitable formulations can be found in formularies known to all pharmacists: Remington's Pharmaceuticals Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (such as LIPOFECTINT™), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., "Compendium of Excipients for Parenteral Formulations," PDA (1998) J Pharm Sci Technol 52:238-311. The composition may contain an antibody or nucleic acid in combination with a medical injection buffer and / or adjuvant.

[0263] The binder polypeptides, or their encoding nucleic acids, can be formulated for the desired route of administration to a patient, for example, as an injectable liquid (optionally an aqueous solution).

[0264] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the present invention. 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. The antigen-binding molecule is preferably administered by subcutaneous injection. Administration may also be self-administration by the patient, e.g., self-injection.

[0265] Pharmaceutical compositions can also be delivered in vesicles, particularly liposomes (Langer (1990) Science 249:1527-1533; Liposomes in the Therapy of Infectious Disease and Cancer, Lopez Berestein and Fidler (eds.), Liss, New York, 1999). See Treat et al., York (1989), pp. 353-365; Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

[0266] In certain circumstances, the pharmaceutical composition can be delivered in a sustained release system. In one embodiment, a pump is used (see Langer, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, a polymeric material is used; Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974). In yet another embodiment, a sustained-release system is placed near the target of the composition, thus requiring only a fraction of the systemic dose (see, e.g., Medical Applications of Controlled Release, Goodson, supra, Vol. 2, pp. 115-138, 1984).

[0267] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injections, infusions, and the like. These injectable preparations can be prepared by known methods. For example, injectable preparations can be prepared by dissolving, suspending, or emulsifying the above-described antibody or its salt in a sterile aqueous or oily medium commonly used for injections. Aqueous media for injection include, for example, saline, isotonic solutions containing glucose, and other auxiliary agents, used in combination with appropriate solubilizers such as alcohols (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. Oily media include, for example, sesame oil, soybean oil, etc., used in combination with solubilizers such as benzyl benzoate, benzyl alcohol, etc. Injectable preparations prepared in this manner can be filled into appropriate ampoules. The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. It is envisioned that the treatment will not be limited to use in a clinic. Thus, subcutaneous injection using a needleless device is also advantageous. With regard to subcutaneous delivery, pen-type delivery devices are easily adapted to deliver the pharmaceutical composition of the present invention. Such pen-type delivery devices may be reusable or disposable. Reusable pen-type delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is emptied, the empty cartridge is easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen-type delivery device can then be reused. In disposable pen-type delivery devices, there is no replaceable cartridge. Conversely, disposable pen-type delivery devices come pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded. Numerous reusable pen-type and autoinjector-type delivery devices are adapted to subcutaneous delivery of the pharmaceutical composition of the present invention. Examples include: Examples of suitable pens include, but are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Burgdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPENT™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIKT™ (Sanofi-Aventis, Frankfurt, Germany), to name just a few. Examples of disposable pen delivery devices adapted for subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, the SOLOSTAR™ pen (Sanofi-Aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly).

[0268] Advantageously, the oral or parenteral pharmaceutical compositions described above are prepared in dosage forms with unit doses suitable for the dosage of the active ingredient. Examples of such unit-dose dosage forms include tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the antibody contained is generally about 5 to about 500 mg per unit-dose dosage form; particularly, in the form of injection, the antibody is contained in an amount of about 5 to about 100 mg, and for other dosage forms, it is contained in an amount of about 10 to about 250 mg.

[0269] The binder polypeptide, nucleic acid, or composition comprising the same is contained in a medical container such as a vial, syringe, IV container, or injection device.In one example, the binder polypeptide, nucleic acid, or composition is in vitro and can be in a sterile container.In one example, a kit is provided that includes the binder polypeptide for use in the therapeutic method described herein, packaging, and instructions for use.

[0270] One aspect of the present invention is a composition comprising a binder polypeptide or nucleic acid of the present invention and one or more pharmaceutically acceptable excipients, examples of which are listed above. "Pharmaceutically acceptable" refers to being approved or expected to be approved by a federal or state regulatory agency for use in animals, including humans, or being listed in the United States Pharmacopoeia or other generally recognized pharmacopeia. A pharmaceutically acceptable carrier, excipient, or adjuvant can be administered to a patient together with a binder polypeptide, e.g., any antibody or polypeptide molecule described herein, does not impair its pharmacological activity, and is non-toxic when administered in a dose sufficient to deliver a therapeutic amount of the drug.

[0271] In some embodiments, the binder polypeptide will be the only active ingredient in a composition according to the invention. Thus, the composition may consist of an antibody, or the composition may consist of a binder polypeptide together with one or more pharmaceutically acceptable excipients. However, a composition according to the invention optionally comprises one or more additional active ingredients. Other therapeutic agents that may be desirable to administer together with a binder polypeptide or nucleic acid according to the invention include other therapeutic agents for iron overload, examples of which are described herein. Any such agent or combination of agents may be administered in combination with a binder polypeptide or nucleic acid according to the invention, or provided in a composition together with a binder polypeptide or nucleic acid according to the invention, whether as a combined formulation or as a separate formulation. A binder polypeptide or nucleic acid according to the invention may be administered separately and sequentially with another therapeutic agent, such as those mentioned, or simultaneously and optionally as a combined formulation. It is possible.

[0272] The compositions can be administered separately or simultaneously. Separate administration refers to two compositions administered at different times, for example, at least 10, 20, 30, or 10-60 minutes apart, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12 hours apart. The compositions can also be administered 24 hours apart or even longer apart. Alternatively, two or more compositions can be administered simultaneously, for example, less than 10 minutes or less than 5 minutes apart. In some embodiments, compositions administered simultaneously are administered as a mixture with or without similar or different time-release mechanisms for each component.

[0273] Binder polypeptides and their encoding nucleic acids can be used as therapeutic agents. The patient herein is generally a mammal, typically a human. The binder polypeptide or nucleic acid can be administered to a mammal, for example, by any of the administration routes described herein. In a preferred embodiment, the binder polypeptide is administered by subcutaneous injection.

[0274] Administration is typically in a "therapeutically effective amount," that is, an amount sufficient to show benefit to the patient and to produce the desired effect for which it is administered. The exact amount will depend on the purpose of the treatment and will be ascertainable by one of skill in the art using known techniques (see, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding). The determination of treatment prescription, e.g., dosage, etc., is within the responsibility of general practitioners and other medical professionals and may depend on the severity of the symptoms and / or progression of the disease being treated. A therapeutically effective amount or appropriate dose of a binder polypeptide or nucleic acid can be determined by comparing its in vitro and in vivo activity in animal models. Methods for extrapolating effective dosages to humans in mice and other test animals are known.

[0275] In the treatment methods described herein, one or more doses are administered. In some cases, a single administration may be effective to achieve long-term benefits. Thus, the method may include a single administration of a binder polypeptide, its encoding nucleic acid, or composition. Alternatively, multiple doses are administered, usually consecutively, and separated by a period of several days, weeks, or months. For example, administration may be every two weeks, three weeks, or four weeks. In some cases, the binder polypeptide is administered to the patient once a month, or less frequently, for example, every two or three months.

[0276] As used herein, the terms "treat," "treating," or "amelioration" refer to therapeutic treatment aimed at reversing, alleviating, improving, inhibiting, slowing, or halting the progression or severity of a condition associated with a disease or disorder. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, 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 the disease is reduced or halted. That is, "treatment" includes not only the improvement of symptoms or markers, but also the cessation, or at least slowing, of the progression or worsening of symptoms compared to that expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), whether detectable or undetectable, diminishment of the extent of the disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, improvement or palliation of the disease state, remission (whether partial or complete), and / or reduced mortality. The term "treatment" of a disease also includes providing relief from the symptoms or side effects of the disease (including palliative treatment). With regard to effective treatment, a complete cure is not contemplated. The methods may be used in certain particular In certain embodiments, it may also include cure. In the context of the present invention, treatment may also be prophylactic treatment.

[0277] A long half-life is a desirable feature of the binder polypeptides of the present invention. A longer half-life leads to less frequent administration, requiring fewer injections to maintain a therapeutically effective concentration of the molecule in the bloodstream. The in vivo half-life of the antigen-binding molecules of the present invention in humans may be 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days or longer. The in vivo half-life of the antigen-binding molecules in non-human primates, such as cynomolgus monkeys, may be 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days or longer.

[0278] Administration of binder polypeptides at regular intervals of one week, two weeks, three weeks, four weeks, or one month is provided.

[0279] therapeutic use Therapeutic applications addressed by the present invention include the treatment of patients who benefit from inhibiting MTP-2 activity and / or reducing iron uptake. A specific therapeutic area is anemia characterized by high iron load and deposition and inadequate erythropoiesis. As previously discussed, some forms of anemia are characterized by ineffective erythropoiesis, often secondary to primary iron overload (caused by inappropriately low hepcidin levels due to high erythropoietic activity) and secondary iron overload, primarily due to repeated red blood cell transfusions. Iron overload contributes to anemia by adversely affecting organ function, negatively affecting erythropoiesis, and increasing hemichrome and ROS in erythroid progenitor cells, driving apoptosis and resulting in few functional red blood cells. Iron reduction reduces apoptosis, improves alpha / beta globin imbalance, and allows for the production of more mature red blood cells. Reduced iron availability to erythroid cells leads to reduced heme production, increased maturation of erythroid precursors, and increased hemoglobin levels. Thus, in some conditions, the therapeutic mechanism of action of binder polypeptides may be the inhibition of the enzymatic activity of MTP-2, which increases the levels of hepcidin, resulting in iron limitation that normalizes erythropoiesis and improves hemoglobin and red blood cell quality.

[0280] In addition to addressing the toxic effects of iron overload, treating anemia may also improve cardiac function and reduce fatigue.

[0281] The effects of treatment according to the present invention include: Reduced absorption of dietary iron, Treatment of iron overload, Increased expression of hepcidin from hepatocytes reducing anemia caused by iron overload, Decreased serum iron concentration, Decreased transferrin iron saturation, reduced need for blood transfusions, reduced need for iron chelation therapy, Prolonged survival, and / or Normalization of red blood cell production.

[0282] Treatment can be beneficial in many conditions, including those discussed herein: Myelodysplastic syndromes (MDS) with lower risk of transfusion-requiring ringed sideroblasts (RARS) 5q-MDS; transfusion-dependent beta-thalassemia or beta-thalassemia major; transfusion-independent beta-thalassemia or beta-thalassemia intermedia; hemochromatosis, or hemochromatosis type 1 or 3, for example, in patients without mutations in HJV or hepcidin; cirrhosis of the liver; fatty liver; For example, NASH (non-alcoholic steatohepatitis) or liver fibrosis in ASH patients; Blackfan-Diamond anemia; pulmonary arterial hypertension; anemia in sickle cell disease ("sickle cell anemia"); polycythemia vera; Chronic kidney disease-related anemia (CKD).

[0283] Thus, the patient to be treated by the present invention may have one of the above conditions. The treatment method may include administering to the patient the binder polypeptide, nucleic acid, or composition described herein. Examples of formulations and administration methods are described elsewhere in this invention. Depending on the disease state, treatment may begin after diagnosis, shortly after birth, or with the onset of transfusion dependence or increased serum ferritin, or otherwise when iron chelation is initiated.

[0284] Increasing hepcidin levels by treatment with anti-MTP-2 binder polypeptides may lead to reduced TSAT levels, accompanied by reduced heme and hemichrome production in beta-thalassemia or reduced ring sideroblast appearance in MDS. This would result in reduced apoptosis of erythroid precursors and higher blood cell counts. Although the hemoglobin content of individual cells would be reduced, total hemoglobin levels would be higher due to the increased red blood cell count. This would then reduce the need for red blood cell transfusions.

[0285] Avoiding or reducing the risk of toxic tissue iron overload should increase overall patient survival, i.e., prolong survival of treated patients. Avoiding or reducing transfusion burden and / or the need for iron chelation and / or phlebotomy should also improve the patient's quality of life.

[0286] Treatment may reduce the disease burden and symptoms of conditions such as beta-thalassemia, MDS, and hemochromatosis that are associated with iron metabolism, including toxic iron overload, heart failure, liver failure, diabetes, and reduced overall survival.

[0287] Interestingly, the Tmprss6 polymorphism 736 V(A)->A(G) has been reported to be associated with higher hepcidin levels and reduce iron overload and improve liver enzymes in some of these diseases. This suggests a positive impact of increased hepcidin levels on liver regeneration. Mouse studies have demonstrated the beneficial effects of increased Tmprss6 and / or hepcidin in mouse liver fibrosis models.

[0288] Inhibition of MTP-2 may also be useful in the treatment of obesity. Folgueras et al. reported that MTP-2 deficiency protects against obesity by regulating iron homeostasis (Folgueras et al., Nat Commun. Apr 10;9(1):1350). 2018).

[0289] Desirably, effective treatment of the patient is obtained without serious adverse effects: no side effects or only mild side effects are observed.

[0290] Treatment with a binder polypeptide according to the present invention can be combined with one or more additional therapies, for example, additional therapeutic agents for treating iron overload. The under polypeptide can be combined with an activin type II receptor agonist fusion protein, such as luspatercept. MTP-2 inhibitors offer an alternative mechanism of action to other planned and existing therapies, potentially providing a synergistic effect on erythropoiesis. The combination of (i) an MTP-2 inhibitor and (ii) a TGFβ superfamily ligand antagonist, such as a TGFβ superfamily receptor-based ligand scavenger / trap, may therefore provide beneficial therapeutic effects.

[0291] Various ligand traps act on the TGFβ superfamily to increase late erythropoiesis. TGFβ superfamily ligands include activin, GDF-11, and bone morphogenetic proteins (BMPs). Receptor ligand trap molecules are created by providing the extracellular domain of a receptor in a soluble form, where the receptor retains the ability to bind its ligand but does not induce downstream signaling (which would otherwise result from normal receptor:ligand interactions). Receptor extracellular domains can be linked to Fc regions to form fusion proteins.

[0292] Preferably, the receptor is an activin II receptor, such as ActIIRA or ActIIRB. The antagonist may also be a polypeptide comprising the soluble extracellular domain of an activin II receptor, such as activin receptor IIB (ActRIIB), fused to an Fc region. Luspatercept is one such molecule, currently used to treat anemia in beta-thalassemia and myelodysplastic syndromes. Examples of ActRIIA and ActRIIB ligand traps can be found in US Pat. No. 7,988,973 to Acceleron Pharma, which is incorporated herein by reference. Suragani RN et al. previously described a modified human ActRIIB extracellular domain (residues 24-131 of the native progenitor with an L79D substitution) linked to a mouse IgG2a Fc domain, "RAP-536," which was reported to reduce ineffective erythropoiesis and disease complications in mouse beta-thalassemia (Blood 123(25):3864-3872, 2014). Sotatercept 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 generated similarly.

[0293] Alternative antagonists include antibodies against TGFβ superfamily ligands (e.g., anti-activin antibodies). Anti-activin A antibodies, such as garetosumab, have been described.

[0294] Antagonists of TGF-β family ligands, such as ActRIIB-Fc, ActIIRIIA-Fc, or BMPR-Fc, promote the maturation of erythroid progenitor cells during erythropoiesis, while MTP-2 inhibitors reduce iron (helping to normalize pathologically high iron levels), thus slowing erythropoiesis. Based on the studies reported herein (see Example 22), the inventors believe that the combination of these two effects results in more efficient production of more mature erythrocytes. For example, the inventors 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, correcting both iron overload and associated anemia. Combination with MTP-2 inhibitors may expand the therapeutic potential of TGF-β superfamily ligand traps by providing greater therapeutic benefit in patients with conditions for which TGF-β superfamily ligand traps are already indicated (e.g., patients with non-transfusion-dependent β-thalassemia). Combination with MTP-2 inhibitors may also expand the therapeutic potential of ligand traps to treat additional patient populations, such as patients with β-thalassemia major, for whom treatments such as luspatercept currently offer limited benefit.

[0295] Luspatercept was recently approved by the FDA for myelodysplastic syndromes. Iron overload begins to occur before MDS patients become transfusion-dependent because ineffective erythropoiesis suppresses hepcidin production in the liver, leading to uncontrolled intestinal iron uptake. Transfusions then exacerbate the iron overload. The same situation occurs in patients with transfusion-dependent β-thalassemia. In such patients treated with luspatercept (or other TGFβ superfamily ligand traps), the inclusion of an MTP-2 inhibitor in the treatment regimen would improve therapeutic benefit. Combination therapy can be used for any of the therapeutic indications or conditions described herein, such as Blackfan-Diamond anemia.

[0296] The inhibitor of MTP-2 used in the combination therapy may be a binder polypeptide as described herein, or may be another type of molecule, such as a nucleic acid inhibitor of TMPRSS6 expression (e.g., an antisense or siRNA molecule targeted to TMPRSS6) or a small molecule inhibitor (e.g., 3-amidinophenylalanine-derived matriptase-1 and -2 protease inhibitors and derivatives). Examples of such inhibitors have been described (Hammami M, Ruhmann E, Maurer E, Heine A, Gutschow M, Klebe G, Steinmetzer T (2012) New 3-amidinophenylalanine-derived inhibitors of matriptase. Med Chem Commun 3:807~813; Pomothy J, Szombath G, Rokonal P, Mathis G, Zs N, Steinmetzer T, Paszti-Gere E (2016) The impact of acute matriptase inhibition in hepatic inflammatory models. Biomed Res Int. https: / / doi.org / 10.1155 / 2016 / 6306984). Thus, patients treated according to the present invention may also be patients receiving treatment with an additional therapeutic agent to reduce iron overload. The method may include co-administering the binder polypeptide and the additional therapeutic agent to the patient, optionally in separate formulations. The compositions may be administered sequentially or simultaneously. The same applies when a small molecule or nucleic acid MTP-2 inhibitor is used instead of the binder polypeptide. That is, the patient is treated either simultaneously or sequentially with a combination of the MTP-2 inhibitor and the additional therapeutic agent. The MTP-2 inhibitor and the additional therapeutic agent are preferably provided in separate formulations and administered separately. Generally, the sequential administration occurs on the same day (optionally separated by a period of minutes or hours) or on different days.

[0297] Treatment with an MTP-2 inhibitor (for example, the binder polypeptide described herein) can be combined with erythropoietin (EPO). Darbepoetin alfa, commercially known as ARANESP, is a structurally reengineered form of EPO to extend the drug's half-life compared with standard EPO alpha and EPO beta proteins. Darbepoetin alfa is used to stimulate erythropoiesis in patients with anemia, increasing hemoglobin levels and reducing transfusion requirements. The drug is administered at different doses depending on the severity of anemia, but the recommended starting dose for patients with chronic kidney disease-related anemia (CKD) is 0.45mcg / kg iv / sc every 4 weeks until hemoglobin levels reach more than 10g / dL, at which point the dose is subsequently reduced. Although ARANESP has been shown to improve hemoglobin levels in a small study in patients with beta-thalassemia intermedia (Singer et al. 2011), ARANESP is generally not a treatment option for beta-thalassemia patients due to an unacceptable increase in RBC apoptosis and accompanying splenomegaly. As the results presented herein demonstrate, therapeutic benefit can be obtained by treating patients with a binder polypeptide according to the present invention and EPO. For example, it should be possible to co-administer ARANESP and anti-MTP-2 mAb treatment in humans by two separate sc injections given simultaneously 2-4 weeks apart. Based on results obtained in a beta-thalassemia mouse model (see Examples 20 and 21), Such a co-treatment strategy should result in therapeutic improvement of anemia from ARANESP therapy while maintaining spleen size and simultaneously reducing toxic iron overload resulting from anti-MTP2 treatment. The exact combined administration regimen will be investigated and optimized in humans, but will generally involve administering a binder polypeptide according to the present invention to a patient, as well as erythropoietin (preferably recombinant erythropoietin or a medically approved variant thereof, e.g., darbepoetin alfa). Optionally, the binder polypeptide and EPO are administered simultaneously. Alternatively, they are administered sequentially (on the same or different days). Optionally, the binder polypeptide and EPO are administered subcutaneously in separate or combined injections. In some embodiments, the present invention provides treatment of a patient with EPO and a binder polypeptide, e.g., in patients with a condition described herein, such as beta-thalassemia, administration of the binder polypeptide reduces red blood cell apoptosis and the associated splenomegaly associated with EPO administration. The binder polypeptides can therefore be used to normalize erythropoiesis in patients undergoing treatment with EPO.

[0298] Next, embodiments of the present invention will be described in more detail with reference to the drawings. [Brief explanation of the drawings]

[0299] [Figure 1]Diagram of pathways controlling iron supply for erythropoiesis and other cellular functions. Dietary iron absorption and the release of iron recovered from senescent red blood cells ("aged RBCs") are controlled by hepcidin, which downregulates ferroportin (FPN), an iron transport protein found in the membranes of intestinal epithelial cells (which take up dietary iron) and macrophages (which take up senescent RBCs). Inhibition of ferroportin reduces iron transport from these cells, thereby limiting the supply of fresh and recycled iron available to the body. Expression of hepcidin from the hamp gene in hepatocytes is controlled by the BMP / SMAD pathway, which is initiated by 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, thereby suppressing ferroportin-mediated iron release. Matriptase-2 (MTP-2) is also present on the hepatocyte surface and downregulates the BMP / SMAD pathway, possibly by cleaving co-receptors such as HJV, thereby reducing hepcidin expression and allowing greater release of iron via ferroportin. [Figure 2] FIG. 1 shows the domain structure of MTP-2. [Figure 3] Graph showing the complete dose-response curves of selected anti-MTP-2 antibodies and a negative control antibody in a protein-based enzyme assay using (a) human and (b) mouse MTP-2. All antibodies are isotype human IgG4PE. [Figure 4] Graph showing the % inhibitory effect of antibodies in an enzyme assay using human MTP-2 ECD plotted against the % inhibitory effect of antibodies in an enzyme assay using mouse MTP-2 ECD for (a) the first and (b) the second antibody groups obtained from transgenic mouse immunization. [Figure 5] 1 is a graph showing the complete dose-response curves of selected anti-MTP-2 antibodies and a negative control antibody in a cell-based enzyme assay using human MTP-2 ECD. All antibodies are of the isotype human IgG4PE. [Figure 6] 1 is a graph showing the results of an HTRF assay of antibodies binding to headless human MTP-2 ECD (recombinant MTP-2 ECD lacking the serine protease domain). IC = isotype control. All antibodies are human IgG4PE. [Figure 7] Graph showing the results of an HTRF assay of antibodies binding to wild-type (a) human, (b) mouse, and (c) cynomolgus MTP-2 ECD. IC = isotype control. All antibodies are human IgG4PE. [Figure 8] Graph showing the results of an HTRF competition assay of selected anti-MTP-2 antibodies or unlabeled aprotinin competing for binding to (a) human and (b) mouse MTP-2 ECD. IC = isotype control for labeled aprotinin-647. All antibodies are human IgG4PE. [Figure 9] Graphs showing (a) hamp mRNA levels and (b) serum iron levels from wild-type mice treated with anti-MTP-2 antibody or control. [Figure 10] Graphs showing (a) hamp mRNA levels, (b) serum iron levels, (c) TSAT, and (d) MCV in wild-type mice treated with NORI-010 10 mg / kg for 24 hours, 7 days, and 14 days, or with a negative control antibody (single time point). [Figure 11] Graphs showing (a) hamp mRNA levels, (b) serum iron levels, (c) TSAT, and (d) MCV in wild-type mice treated with NORI-010 3 mg / kg for 24 hours, 7 days, and 14 days, or with a negative control antibody (single time point). [Figure 12] Graphs showing (a) hamp mRNA levels and (b) serum iron levels from mice treated with human or mouse anti-MTP-2 antibodies or their isotype controls. [Figure 13]Graphs showing the results of a time course evaluation of NORI-008 in terms of (a) hamp, (b) serum iron, and (c) PK following a single ip injection of 10, 3, and 1 mg / kg into normal mice. [Figure 14] 1 is a graph showing the results of a one-week evaluation of an IgG / kappa anti-MTP2 antibody (10 mg / kg IP dose) for reducing serum iron and transferrin saturation. [Figure 15] 1 is a graph showing the results of an evaluation of three anti-MTP-2 antibodies of fully human IgG4 and mouse IgG1 configurations for their ability to reduce serum iron levels after subcutaneous injection. [Figure 16] Graphs showing (a) serum iron concentrations and (b) antibody concentrations 7 days after a single sc or ip injection of 10 mg / kg of antibody. In (a), black circles represent the huIgG4PE isotype control and gray squares represent NORI-010. [Figure 17] 1 is a graph showing the results of a first in vivo rat study in which NORI-008 and NORI-010 were dosed IP at 10 mg / kg to groups of three wild-type Wistar rats. [Figure 18-1] 1 is a graph showing the results of a second in vivo rat study in which NORI-008, NORI-011, NORI-003, and NORI-006 were each dosed SQ at 10 mg / kg to groups of five wild-type Han Wistar rats. [Figure 18-2] Continuation of Figure 18-1. [Figure 19] Graphs showing the results of a 2-week study of NORI-010 in the Hbbth3 / + mouse model of thalassemia intermedia: a) hepcidin mRNA from liver samples measured by qPCR, b) serum iron levels [μg / dL] measured by chromogenic assay, and c) calculated transferrin saturation [%]. [Figure 20-1]Graphs showing the results of an 8-week study in the Hbbth3 / + mouse model of thalassemia intermedia using NORI-011-M in the presence or absence of erythropoietin: a) hepcidin mRNA from liver samples measured by qPCR, b) red blood cell count, c) hepatic iron content per gram of wet tissue, 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 erythroid developmental stages I-V cells in the spleen determined by flow cytometry, j) number of erythroid developmental stages I-V cells in the bone marrow determined by flow cytometry, and k) spleen index. [Figure 20-2] Continuation of Figure 20-1. [Figure 21-1] Figure 1 shows the results of an 8-week study in the Hbbth3 / + mouse model of thalassemia intermedia using NORI-011-M with or without co-treatment with ActRIIB-Fc. NORI-011-M was administered ip at 10 mg / kg once weekly, and ActRIIB-Fc was administered ip at 10 mg / kg twice weekly. All samples for readout were obtained at the end of the 8-week study. a) Hepcidin mRNA from liver samples measured by qPCR, b) liver iron tissue content per gram of wet tissue, 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) number of erythroid developmental stages I-V cells in the spleen determined by flow cytometry, j) number of erythroid developmental stages I-V cells in the bone marrow determined by flow cytometry, and k) spleen index. [Figure 21-2] Continuation of Figure 21-1. [Figure 21-3] Continuation of Figure 21-2. [Example]

[0300] We describe here antibodies targeting MTP-2 for the treatment of iron overload diseases. By immunizing transgenic mice that generate antibodies with human variable domains and testing a wide variety of antibodies in a series of biologically relevant assays, we were able to obtain species-crossreactive MTP-2-specific monoclonal antibodies (mAbs) that neutralize MTP-2 enzymatic activity both in vitro and in vivo. We demonstrate that selected mAbs increase hepcidin expression levels from liver cells after a single dose. Elevated hepcidin reduces serum iron and transferrin saturation by increasing the internalization and degradation of ferroportin. Hbb, a model of beta-thalassemia, is a common marker for hemoglobin-dependent iron deficiency. th3 / + In mice, a single dose of 10 mg / kg resulted in a 52% and 47% reduction in serum iron and transferrin saturation, respectively, at 2 weeks. Furthermore, with repeated dosing, the inventors demonstrated a significant reduction in Hbb th3 / + Consistent iron restriction was observed in mice over multiple weeks. These results indicate that such mAbs have the potential to treat iron-overloaded patients, reducing their anemia and the need for blood transfusions and iron chelation. Example 1

[0301] Generation of a panel of anti-MTP-2 inhibitor antibodies Kymab transgenic mice, which produce antibodies with human variable domains, were immunized with MTP-2 using a variety of different immunization regimens and antigen configurations, and antigen-specific B cells were selected. See Lee et al., Nat Biotechnol 32(4):356-63 2014; WO2011 / 004192; WO2011 / 158009, and WO2013 / 061098. The 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 protein- and cell-based in vitro assays.

[0302] Homogeneous time-resolved FRET (HTRF) and flow cytometry assays were used as primary screens to demonstrate binding of the recovered antibodies to purified MTP-2 extracellular domain (ECD), followed by confirmation of binding to cell surface-expressed MTP-2.

[0303] Cross-reactive antibodies were selected for their ability to bind to both human and non-human (mouse and cynomolgus monkey) MTP-2 ECD.

[0304] Selected antibodies were then screened in several functional assays to assess their ability to inhibit the enzymatic activity of human and mouse purified MTP-2 ECD in solution, as well as the enzymatic activity of human MTP-2 expressed in HEK293 cells, as assessed in an enzymatic assay containing a chromogenic MTP-2 substrate.

[0305] Although many different antibodies were obtained, including examples of antibodies that bound to and inhibited human MTP-2 but did not bind to or inhibit mouse MTP-2, and other antibodies that bound to and inhibited mouse MTP-2 but did not bind to or inhibit human MTP-2, the antibodies listed in Table G below were selected as being particularly interesting in terms of their potential for development as cross-reactive inhibitors of MTP-2 activity.

[0306] [Table 1] Example 2

[0307] Antibody sequence 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 for each antibody is shown. The complete light chain for each antibody is also shown. Unless the context indicates otherwise, "NORI-001" refers to the antibody having the VH and VL domains shown in Table S as NORI-001. The composition of the antibody may also be indicated, for example, "NORI-001 IgG" is an IgG having the NORI-001 VH domain and the NORI-001 VL domain, and "NORI-001" is an IgG having the NORI-001 VH domain and the NORI-001 VL domain. "scFv" is an scFv having the NORI-001 VH domain and the NORI-001 VL domain.

[0308] Antibody NORI-002 was identified as having a high-risk free cysteine ​​liability in the VH domain. This amino acid, which was considered to be filled by structural software analysis, was mutated to alleviate any potential liability. Therefore, a C49C mutation was introduced into NORI-002, and the new antibody containing this mutation was designated NORI-003.

[0309] Antibody NORI-010 was sequence optimized by introducing a P124S mutation into the VH domain to improve stability and expression, thereby generating a new antibody, NORI-011. Example 3

[0310] Inhibition of MTP-2 in a protein-based enzyme assay using the MTP-2 ECD The antibodies were evaluated for their ability to inhibit serine protease cleavage of a labeled MTP-2 substrate to generate a detectable product in an enzyme assay using human and mouse MTP-2 ECD.

[0311] Antibodies NORI-001 through NORI-034 all inhibited the enzymatic activity of both human and mouse MTP-2 in this assay. The IC50 values ​​for inhibition of human MTP-2 ranged from approximately 1.5 to 55 nM. The IC50 values ​​for inhibition of mouse MTP-2 ranged from approximately 0.48 to 40 nM. Table D. Figure 3.

[0312] [Table 2-1] [Table 2-2]

[0313] Antibody cross-reactivity was assessed by comparing inhibition in assays using human MTP-2 ECD with inhibition in assays using mouse MTP-2. By plotting the percentage inhibition values ​​in the two assays against each other, it was possible to observe that some antibodies were specific for human MTP-2, while others were specific for mouse MTP-2, and that there were various degrees of inhibition against both human and mouse MTP-2 (Figure 4).

[0314] Materials and Methods for Protein-Based Enzyme Assays In the initial assay, the positive and negative controls, aprotinin (Sigma - A3428) and a non-MTP-2 binding huIgG4PE isotype antibody, respectively, were serially diluted 1:3 into assay buffer (200 mM Tris-HCl and 1 mg / mL BSA, pH 9.0) at a starting concentration of 200 nM at 2x final concentration. In subsequent assays, the positive and negative controls used were NORI-008 huIgG4PE and a non-MTP-2 binding huIgG4PE isotype antibody at 10 nM, respectively.

[0315] Twenty microliters of titrated control and diluted antibody were plated into a 384-well solid white plate (Alpha plate - 6005350), and 10 μl of human or mouse MTP-2 ECD protein was plated on top at a final concentration of 0.5 or 2 mg / ml, respectively, for enzyme activity differences. The plate was then covered and incubated at room temperature for 30 minutes. Ten microliters of the 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 μM in assay buffer. The enzyme reaction was allowed to proceed at room temperature, and fluorescence activity was then read at 30 minutes, 1 hour, and 2 hours in a plate reader (Envision) at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. Upon cleavage of the substrate peptide via the enzymatic activity of the MTP-2 enzyme, a 7-amido-4-methylcoumarin (AMC, MCA, or NHMec) moiety is released from the C-terminus. AMC is a fluorophore incorporated into the carboxypeptidase substrate at the C-terminus. The released coumarin can then be excited at 360–380 nm using a spectrofluorometer, with emission at 440–460 nm detected. Positive and negative controls were obtained as the average of 16 wells of aprotinin / NORI-008 IgG and huIgG4PE isotype, respectively, at a final concentration of 200 nM. Inhibition values ​​were calculated using GraphPad software. Logarithmic curves were generated by inputting data into the software, and IC50 values ​​were generated using nonlinear regression parameters and a log (inhibition) vs. response-variable slope (four parameter) equation.

[0316] See Example 5 for details on preparation of antigen reagents. Example 4

[0317] Inhibition of MTP-2 in a cell-based enzyme assay using cell surface-expressed MTP-2 The antibodies were evaluated for their ability to inhibit serine protease cleavage of a labeled MTP-2 substrate to generate a detectable product in an enzyme assay using cell surface-expressed human MTP-2.

[0318] 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 in this assay. The IC50 values ​​for inhibition of human MTP-2 ranged from approximately 0.083 nM to 17 nM. Table D. Figure 5.

[0319] In this cell-based assay, MTP-2 is expressed by cells and displayed on the cell surface where it undergoes autoactivation to produce activated MTP-2, which cleaves the substrate. Inhibitors in this assay can act by various molecular mechanisms. For example, inhibitors that show inhibition in this assay may bind to the MTP-2 zymogen and prevent its conversion to autoactivated MTP-2 (Figure 2), thereby preventing the formation of the activated form of MTP-2 that would otherwise cleave the substrate in this assay, and / or inhibitors in this assay may bind to and inhibit the activated form of MTP-2.

[0320] The ability of antibodies NORI-001 through NORI-034 to inhibit cell surface-expressed MTP-2 confirms their activity against MTP-2 in a cell-based setting where the target antigen is expressed and activated on cells, constituting an in vivo situation.

[0321] Materials and methods for cell-based enzyme assays All antibodies for screening, as well as positive and negative controls, were serially diluted 1:3 in Expi293 medium (A1435101) (serum-free medium supplemented with high glucose and GlutaMAX, pH 8) at a starting concentration of 200 nM for 2x final concentration. 12.5 μl of antibody (Protein A purified) and control were plated into a 96-well solid white plate (Corning - CLS3917-100EA), and 25 μl of cells in Expi293 medium (6250 cells per well) were dispensed on top. Then, 12.5 μl of fluorescent MTP-2 peptide substrate (Bachem AG - 4016429.0050) was added to each well at a final concentration of 50 μM in Expi293 medium. The enzymatic reaction was allowed to proceed overnight in an incubator at 37°C and 5% CO2. The next day, fluorescence activity was read in a plate reader (Envision) at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. Positive and negative controls were taken as the average of four wells of aprotinin and huIgG4PE isotype at a final concentration of 200 nM, respectively.

[0322] In initial assays, the positive control was aprotinin (Sigma - A3428). In subsequent assays, the positive control was NORI-008 huIgG4PE. A non-MTP-2 binding huIgG4PE isotype antibody was used as a negative control.

[0323] See Example 5 for details on preparation of antigen reagents. Example 5

[0324] Production of antigenic material Generation of constructs for protein expression To generate purified proteins for use in the assays described herein, Extracellular domain (ECD) aa77-855 of WT human matriptase-1 (uniprot sequence number Q9Y5Y6) Wild-type (WT) human MTP-2 (uniprot sequence number Q8IU80) aa 78-811 WT mouse MTP-2 (uniprot sequence number Q9DBI0) aa80-811 WT rat MTP-2 (NCBI sequence number XP006242057.1) aa80-811 Cynomolgus monkey MTP-2 (uniprot sequence number A0A2K5VAP0) aa 73-800 The DNA sequence encoding the

[0325] The above amino acid sequences are also provided in Table S for reference.

[0326] These coding sequences were fused with a C-terminal His tag and an N-terminal leader sequence, codon-optimized for mammalian expression, and expressed.

[0327] To generate the MTP-2 antigen, coexpression with the untagged moHAI-2 ECD antigen (followed by purification of the His-tagged antigen through a nickel column) significantly improved expression. To this end, DNA encoding WT mouse HAI-2 (uniprot sequence number Q9WU03) aa 28-197 was fused to an N-terminal immunoglobulin leader sequence, codon-optimized for mammalian expression, and expressed.

[0328] The DNA sequence was cloned into the pTT5 protein expression vector under the control of the CMV promoter using the Golden Gate method and the AarI restriction site. The expression plasmid was transfected into CHO-3E7 cells using PEI transfection reagent.

[0329] Generation of full-length antigen constructs for stable cell line generation To screen for MTP-2-specific antibodies, we generated stable cell lines expressing the relevant antigen.

[0330] The full-length DNA sequences encoding wild-type (WT) human MTP-2 (uniprot SEQ ID NO: Q8IU80) amino acids (aa) 1-811 and WT mouse MTP-2 (uniprot SEQ ID NO: Q9DBI0) aa 1-811, fused with an N-terminal eGFP and a C-terminal flag tag (DYKDDDDK), respectively, were codon-optimized for mammalian expression. This 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 flanked by 3' and 5' piggyBac-specific terminal repeats to facilitate stable integration into the cellular genome (see "A hyperactive piggyBac transposase for mammalian applications"; Yusa K. et al., Proc. Natl. Acad. Sci. USA., 108(4):1531-6, January 25, 2011). The CMV promoter expression vector contained a puromycin selection cassette to facilitate stable cell line generation.

[0331] WT human MTP-2 aa 10–811 and WT mouse MTP-2 amino acids 13–8 Isoform 2, i.e., untagged versions of 11, were generated from the above constructs by PCR-directed mutagenesis and recloned into the same expression vector as before. Isoform 2, i.e., untagged versions of the K253E, V736A, and K253E+V736A human WT MTP-2 (uniprot sequence number Q8IU80) sequence aa10-811 variants were also generated by PCR-directed mutagenesis and recloned into the same expression vector as before.

[0332] The full-length DNA sequences encoding wild-type human matriptase-1 (MTP-1) (uniprot SEQ ID NO: Q9Y5Y6) aa 1-855 with a C-terminal His-tag fusion, wild-type human matriptase-3 (MTP-3) (uniprot SEQ ID NO: Q7RTY8) aa 1-854 with a C-terminal His-tag fusion, and untagged wild-type mouse HAI-2 (uniprot SEQ ID NO: Q9WU03) aa 1-252 were all codon-optimized for mammalian expression and cloned into the same expression vectors with a CMV promoter as before. The expression vectors for MTP-1 and MTP-3 contained a puromycin selection cassette, and the expression vector for moHAI-2 contained a neomycin selection cassette to facilitate dual stable cell line generation.

[0333] Generation of stably transfected Hepa1-6, CHO, and HEK293 cells expressing MTP-1, MTP-2, and MTP-3 antigens For the generation of a human embryonic kidney (HEK) 293 cell line expressing WT human MTP-2 eGFP / Flag-tagged aa 1–811, a CMV promoter expression plasmid was cotransfected 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.

[0334] For the generation of Chinese hamster ovary (CHO) cell lines expressing WT human MTP-2 eGFP / Flag-tagged aa 1–811, a CMV promoter expression plasmid was cotransfected with a plasmid encoding piggyBac transposase into Chinese hamster ovary (CHO) cells using FreeStyle Max transfection reagent (Invitrogen) according to the manufacturer's instructions.

[0335] To generate cell lines expressing untagged WT human MTP-2 aa10-811 and WT mouse MTP-2 aa13-811, a CMV promoter expression plasmid was cotransfected with a plasmid encoding piggyBac transposase into HEK293 cells. The WT human aa10-811 and WT mouse aa13-811 MTP-2 untagged constructs were also cotransfected with a plasmid encoding piggyBac transposase into the Hepa1-6 cell line using FreeStyle Max transfection reagent (Invitrogen) according to the manufacturer's instructions.

[0336] For the generation of cell lines expressing His-tagged WT human MTP-1 aa10–855 and WT human MTP-3 aa1–854, MTP-1 and MTP-3 CMV promoter expression plasmids were cotransfected into the HEK293 cell line together with a WT moHAI-2 expression plasmid and a plasmid encoding piggyBac transposase using FreeStyle Max transfection reagent (Invitrogen) according to the manufacturer's instructions.

[0337] 24 hours after transfection, the medium was supplemented with puromycin (2.5 μg / mL) or G418 (1 mg / mL), or puromycin (2.5 μg / mL) and G418 (1 mg / mL). Stable cell lines were selected by supplementing the medium with both 18 (1 mg / mL) and growing for at least two weeks. The cell culture medium was changed every 3–4 days. After selection, the WT human MTP-2 eGFP / Flag-tagged aa1–811-expressing HEK293 cell line was serially diluted to a monoclonal cell line for highest expression. This was repeated for the WT and S762A human MTP-2 eGFP / Flag-tagged aa1–811-expressing CHO cell lines. Cellular expression of flag-tagged human or mouse MTP-2 constructs was assessed by flow cytometry using an anti-flag APC-conjugated antibody (Biolegend - 637308), and expression of his-tagged human MTP-1, human MTP-3, and human and mouse MTP-2 constructs was assessed by flow cytometry using an unlabeled anti-His primary antibody (Abcam - ab18184) followed by a goat anti-mouse 647-conjugated secondary antibody (Citeab - 115-605-071). After selection, stable untagged WT human MTP-2- and WT mouse MTP-2-expressing HEK293 cells and stable untagged WT human MTP-2- and WT mouse MTP-2-expressing Hepa1-6 cells were FACS sorted for high expression. Cellular expression of untagged human, mouse, and cynomolgus MTP-2 constructs, including human and mouse mutant and human variant constructs, was assessed by flow cytometry using an APC-conjugated anti-MTP-2 antibody.

[0338] HEK293 and Hepa1-6 complete medium consisted of Dulbecco's Modified Eagle's Medium (Gibco) supplemented with 10% v / v fetal bovine serum (Gibco). During transfection, adherent CHO cells were cultured in Ham's F-12 Nutrient Mix (Gibco) supplemented with 10% v / v fetal bovine serum (Gibco). After transfection, antigen-expressing CHO cells were cultured in suspension in CHO-S complete medium, which consisted of CD-CHO medium supplemented with 8 mM Glutamax (Gibco). The CHO-3E7 cells used here for expression contained the pTT5 vector system and CHO EBNA 1 cells available from the National Research Council of Canada, although other CHO cell lines may also be used. Example 6

[0339] Surface plasmon resonance determination of binding affinity and kinetics Dissociation rate screening was performed by surface plasmon resonance (SPR) using a Biacore 8K system (GE Healthcare). Anti-human Fc mix (approximately 1000 RU) was immobilized in both the active and reference channels in HBS-P+ buffer (GE BR100671) at pH 7.4. Anti-MTP-2 huIgG4PE antibody was then captured at 10 μl / min for 60 s at a concentration of 1 μg / ml in the active channel only (approximately 35 and 50 RU were captured). MTP-2 ECD protein analyte was then injected at 30 μl / min for 120 s (association time) (at concentrations of 100, 25, 6.25, 1.56, and 0 nM), and dissociation was monitored for 600 s. Multicycle kinetic analysis was used for all eight channels. Reference and background were subtracted from each antibody sensorgram, and the data were fitted using a 1:1 interaction model in the Biacore evaluation software. Rmax, ka, globally fitted kd, RI=0.

[0340] Affinity (K D ) ranged from approximately 0.012 nM to 8.5 nM. Table K.

[0341] [Table 3]

[0342] We also evaluated binding using the SPR method described above using the "headless" huMTP-2 ECD 78-576aa instead of the complete 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 shown in Table S. No binding to the headless protein was detected for any of the seven antibodies, suggesting that all epitopes for these antibodies reside in the serine protease domain.

[0343] [Table 4]

[0344] NORI-011, NORI-008, NORI-003, and NORI-006 were all found to have cross-reactive binding to human, mouse, cynomolgus monkey, and rat MTP-2 ECD proteins. The sequences of these proteins used in SPR are listed in Table S as follows: human matriptase-2 ECD protein his-tag, mouse matriptase-2 ECD protein his-tag, cynomolgus monkey matriptase-2 ECD protein his-tag, rat matriptase-2 ECD protein his-tag, and human matriptase-2 mask ECD protein his-tag, respectively. The KD values ​​of NORI-008 could not be accurately determined for either human or mouse MTP-2 because of the very slow dissociation rates. No binding to the human matriptase-2 masked ECD protein his-tag was detected for any of these four antibodies, indicating that binding was not to the his-tag and that the epitopes of all antibodies reside in the serine protease domain. Example 7

[0345] Binding to the MTP-2 serine protease domain SPR analysis performed on the panel of antibodies in Example 6 showed that no binding was detected to a truncated, "headless" huMTP-2 protein lacking the serine protease domain, suggesting that these antibodies bind to the serine protease domain of MTP-2. Such antibodies are predicted to mechanistically block MTP-2 by blocking or distorting the serine protease domain active site, preventing substrate cleavage.

[0346] HTRF binding assays of NORI-003, NORI-006, NORI-008, and NORI-011 against this headless protein confirmed the absence of detectable binding (Fig. 6), whereas binding was detected against all human, mouse, and cynomolgus MTP-2 proteins without C-terminal truncations (Fig. 7). This also suggested that the binding epitopes of these inhibitory antibodies reside in the serine protease domain. Another antibody, NORI-036, bound to both the headless and complete ECDs, indicating that NORI-036 recognized a binding site on MTP-2 outside the serine protease domain. Example 8

[0347] Competition with aprotinin Aprotinin is a pan-serine protease inhibitor known to occupy the active site of serine proteases. HTRF competition assays against labeled aprotinin confirmed that NORI-003, NORI-006, NORI-008, and NORI-011 bound to or near this same site and competed with aprotinin for binding to human MTP-2 protein. For NORI-003 and NORI-006, this competition was weak despite their large enzyme inhibition IC50 values ​​against huMTP-2, suggesting that the epitopes of these clones were likely near the active site but not identical to the binding site of aprotinin. This was confirmed by the lack of competition between NORI-003 and NORI-006 and aprotinin for moMTP-2, despite their IC50 values ​​in the enzyme assay being comparable to that of NORI-011. NORI-008 exhibited very low Kd and IC50 values ​​in the SPR and aprotinin competition assays, respectively, suggesting that its binding site was identical to or closely overlapped with that of aprotinin (Figure 8, Table L).

[0348] [Table 5]

[0349] Materials and methods for HTRF competition assay Antibodies were titrated in 4x HTRF buffer (DPBS (Gibco - 14190144) containing 0.1% BSA (Sigma - A7906) and 0.53M potassium fluoride (Sigma - 60240-250G)) at a starting concentration of 200 nM. 5 μL / w of antibody was added to a 384-well white plate (Greiner - 784904). Purified proteins of huMTP-2 and moMTP-2 were diluted in HTRF buffer to 4x final concentrations (huMTP-2 = 40 nM and moMTP-2 = 240 nM) and plated at 5 μL / w. Next, a moIgG1-based anti-MTP-2 mAb (NORI-037) was diluted in HTRF buffer to a 4x final concentration of 1.2 nM along with a 1:1000 dilution of DELFIA Eu-N1 rabbit anti-mouse IgG antibody (AD0207) at a 4x final concentration. Finally, 647-labeled aprotinin (Sigma - A3428) was diluted in HTRF buffer to a 4x final concentration of 20 nM and plated at 5 μL / w. Plates were incubated in the dark at RT for 3 hours or longer. HTRF Plates were read at 1, 2, and 3 hours in an EnVision plate reader using a 100 flash protocol (Ex: 340 nm, Em1: 620 nm, Em2: 665 nm). Example 9

[0350] Progress towards in vivo studies MTP-2 has been recognized as a target for addressing iron overload anemia by increasing hepcidin concentrations through various models and concepts. Absence or blocking of MTP-2 activity has been shown in human genetics, preclinical models, and clinical interventions to increase hepcidin levels and thus reduce iron overload. For example, Tmprss6 knockout mice are viable but characterized by high hepcidin levels and, therefore, iron restriction, which results in a transient so-called "mask" phenotype that causes hair loss on the body but not on the head. Crossing these mice with beta-thalassemia mice with hemizygous beta-globin chain loss results in the development of the phenotype seen in these crosses. Due to increased hepcidin, red blood cell counts and hemoglobin can improve (Nai et al., 2012). Furthermore, in humans, mutations in the Tmprss6 gene cause abnormally high hepcidin levels, resulting in a rare form of anemia in which patients suffer from iron deficiency (iron-refractory iron deficiency anemia - IRIDA) that cannot be corrected by providing more iron (Lenoir et al., Blood 117:647-650 2011; Nai et al., 2012, supra).

[0351] Inhibition of MTP-2 activity indicates that the antibody can increase hepcidin expression and thus prevent iron overload, a major cause of morbidity and mortality in beta-thalassemia. Therefore, antibodies with such activity confirmed in vivo may be able to ameliorate anemia in beta-thalassemia and may be useful therapeutic agents for treating patients with or at risk of iron overload.

[0352] In vivo evaluation included assessing increased hepcidin gene mRNA transcription from liver cells and reduced serum iron in wild-type mice. Readouts were obtained within 24 hours of dosing, allowing rapid confirmation of antibody activity.

[0353] The desired mechanism of action is the inhibition of WT MTP-2 enzymatic activity and the cleavage of downstream substrates at the cell surface of liver cells. The immediate effect of this inhibition is a 2- to 8-fold increase in hepcidin mRNA transcription, observable within 6 hours of administration. The increased hepcidin expression leads to the suppression of serum iron and, therefore, transferrin saturation, within a comparable time frame. In healthy animals, continued MTP-2 inhibition and serum iron restriction result in reduced mean corpuscular volume (MCV) and red blood cell distribution (RDW), which typically begin to manifest after 2 weeks if the drug remains present and active.

[0354] Antibodies selected for in vivo testing were chosen based on their concentration-dependent inhibition of enzymatic activity for human and mouse MTP-2 protein and cell surface-expressed MTP-2. Antibodies were excluded if they were deemed to have potential difficulties with expression and / or purification yield, posed a risk to their developability, did not show complete enzymatic inhibition in vitro for either the human or mouse ECD protein, or generated IC50 values ​​that were deemed too low.

[0355] Two antibodies that showed weak inhibition in the enzyme assay 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 enzyme activity to function well in vivo. Therefore, in vitro inhibition is considered a necessary criterion for in vivo efficacy. However, it is likely not necessarily a sufficient condition for achieving in vivo efficacy. For some antibodies, we observed in vivo performance that was strong in the short term but was not maintained over time. Other factors, such as pharmacokinetics or anti-drug antibodies generated by mice, may affect longer-term in vivo performance in these models. Example 10

[0356] Protocol for determining the effects of anti-MTP-2 antibodies on hepcidin mRNA and serum iron in wild-type mice Iron Assay Protocol To generate the data shown in Figures 13, 14, 15, 16, 17, and 18, iron quantification was performed using the QuantiChrom™ Iron Assay Kit (Bioassay System, DIFE-250).

[0357] Briefly, iron standards were prepared according to the kit's protocol. After this, 25 μl of standard or sample was added to the wells of a 96-well plate, followed by 200 μl of Reagent A. The plate was then read at 595 nm in a microplate spectrophotometer (Reading A). 10 μl of Reagent B was then added to the wells, followed by 10 μl of Reagent C. The plate was then incubated at room temperature for 40 minutes and read at 595 nm in a plate reader (Reading B). The increase in absorbance for all wells was calculated by subtracting Reading A from Reading B. A standard curve was then plotted, and unknown values ​​of sample iron were read from the standard curve.

[0358] To generate the data shown in Figures 9, 10, 11, 12, and 19, serum iron content analysis was performed using the Direct Iron Assay (Ferene) kit according to the protocol supplied in the kit.

[0359] Briefly, 25 μL of serum from standard or research animals was incubated with 125 μL of solution R1 and 25 μL of solution R2 in 96-well plates, and then the plates were read in the envision microplate reader at 600 nm according to the CLF protocol (absorbance A1). 2.5 μL of chromogen was added. After 20 minutes of incubation at room temperature, the plates were read according to the same envision protocol as before (absorbance A2). The results were:

number

[0360] Any value calculated as less than 0 is reported as 0.

[0361] Transferrin saturation (TSAT) calculation TSAT analysis was carried out using the iron fixation latency measurement kit according to the protocol provided in the kit. Briefly, 25 μL of serum from standard or research animal was incubated with 125 μL of solution R1 in a 96-well plate at room temperature for 3 minutes. The plate was then read in an envision microplate reader based on the 600 nm CLF protocol (absorbance A1). 25 μL of serum from standard or research animal was incubated with 125 μL of standard solution (volume of R1:R2 50:1) at room temperature for 5 minutes. The plate was then read based on the same envision protocol as before (absorbance A2). The results are:

number

[0362] Any value calculated as less than 0 is reported as 0.

[0363] Analysis of serum antibody levels The following assay was used to confirm serum antibody concentrations: 96-well plates were coated with 50 μL / well of mouse anti-human IgG4 Fc (2 μg / mL in PBS) overnight at 4°C. The plates were washed three times with 300 μL / well of PBD-T (PBS with 0.1% Tween) using a plate washer. The plates were blocked for 1 hour at room temperature using 150 μL / well of PBS supplemented with 1% BSA. Samples were diluted (using pooled mouse serum) to prepare QCs and standard curves (10-step series, 7.81–2000 ng / ml). The plates were then washed three times with 300 μL / well of PBD-T (PBS with 0.1% Tween) using a plate washer. 50 μL per well of the standard curve, sample, or QC was added to the assay plate, and the assay plate was then incubated at room temperature for 1 hour with shaking at 300 RPM. The plates were then washed three times with 300 μL / well of PBD-T (PBS with 0.1% Tween) using a plate washer. 50 μL / well of HRP-conjugated mouse anti-human kappa diluted 1:12,000 in PBS with 1% BSA was added to the plate, which was then incubated at RT for 1 hour with shaking at 300 RPM. The plate was then washed three times with 300 μL / well of PBD-T (PBS with 0.1% Tween) using a plate washer. 100 μL of TMB substrate was added to each well. The plate was incubated at RT for 10 minutes in the dark. 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 at 450 nm, with a reference reading above 540 nm. The reference reading was subtracted from the reading at 450 nm. The data was then imported into Softmax Pro, and regression was performed using 4PL curve fitting with a weighting factor of 1 / y for the standard of the sample concentrations read from the standard curve.

[0364] RNA extraction RNA was prepared from liver samples using the Qiagen RNeasy Plus Mini Kit according to the kit's protocol. Frozen samples were thawed on wet ice. 600 μl of Buffer RLT Plus was then added to the sample. The sample was then homogenized using a plastic pestle. The sample was then tritiated using a 1 ml syringe and a 20G needle. The sample was then centrifuged, and the supernatant was placed in a gDNA removal spin column in a 2 ml Eppendorf tube, and the pellet was discarded. The spin column and tube were centrifuged, retaining the flow-through, and the column was discarded. 600 μl of 70% ethanol was then added to the flow-through. 700 μl of sample was then loaded onto an RNeasy Spin Column placed in a 2 ml collection tube and then centrifuged. The flow-through was discarded. The spin column was then returned to the 2 ml collection tube. 700 μl 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 returned to the 2 ml collection tube, 500 μl of Buffer RPE was added to the spin column, and the spin column was centrifuged. The flow-through was discarded, and washing the column with Buffer RPE was repeated as above. Centrifuged at 8000 × g for 15 seconds. The spin column was placed in a new 2 ml collection tube and centrifuged for 1 minute at maximum to dry the membrane. The spin column was placed in a new 1.5 ml collection tube, and 30–50 μl 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 RNA levels in the flow-through were estimated using a nanodrop (see below), and the solution was then stored at -20°C or -80°C.

[0365] qPCR analysis After mRNA extraction, mRNA from each mouse liver was quantified using nanodrop and normalized to 5 ng / μl. Briefly, transcript levels of mouse hepcidin (hamp) mRNA were measured by qRT-PCR and normalized to the mouse hypoxanthine-guanine phosphoribosyltransferase (HPRT) mRNA housekeeping gene. Five μl of mRNA extract (25 ng total) was then mixed with 10 μl of QuantiTect Probe RT-PCR Kit and 20X hamp FAM probe mix. 1 μl of 20X HPRT VIC probe mix, 1 μl of 40X TaqMan 0.5 μl of RT enzyme mix was mixed with 2.5 μl of RNA-free HO to a final volume of 20 μl in a 96-well semi-skirted qRT-PCR plate. The setup for the qRT-PCR reaction included a 15-minute reverse transcription step at 48°C, followed by a 10-minute activation step at 95°C, followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. The -ΔCt value was then calculated by subtracting the Ct value of Hamp from that of HPRT. Example 11

[0366] 24-hour single-dose evaluation of four IgG / lambda anti-MTP2 antibodies in normal mice for reducing serum iron and transferrin saturation NORI-009, NORI-010, NORI-012, and NORI-034 were included in the initial evaluation in healthy mice as fully human IgG4λ mAbs. Nine-week-old C57BL / 6 male mice were administered one intraperitoneal antibody injection at 10 mg / kg (150 μl / mouse), five mice per group. Mice were sacrificed 24 h after injection. As a positive control for hepcidin induction, three 9-week-old C57BL / 6 male mice were injected with LPS (1 μg per gram of body weight) and sacrificed 4 h later. LPS (lipopolysaccharide from Escherichia coli) mimics bacterial infection, so mice respond by inducing hepcidin expression and reducing serum iron. LPS serves as a positive control for agents known to induce an acute inflammatory response and induce maximal hepcidin increases based on the inflammatory response. Real-time PCR for hamp, Id1, 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 from all mice, and transferrin saturation was calculated.

[0367] NORI-034 was not found to increase hepcidin expression above the levels seen in isotype control-treated mice. On the other hand, NORI-009, NORI-010, and NORI-012 all increased hepcidin expression by at least 1 ct value above the mean of the isotype control-treated group 24 hours after dosing and equaled the hepcidin expression induced by the LPS positive control 4 hours after dosing. As a result, serum iron levels in mice treated with these antibodies were reduced to an average of less than 40 μg / dL. (Figure 9)

[0368] This experiment demonstrated the biological relevance of MTP-2 to the BMP / SMAD / hepcidin pathway and showed for the first time that antibody-directed inhibition can reduce serum iron in normal mice. Because NORI-034 was considered inactive in vivo, this antibody was not explored further. Example 12

[0369] Time course evaluation of NORI-010 after a single ip injection in normal mice Antibody NORI-010 was one of the antibodies that showed a favorable profile in Example 11, and therefore it was of interest to determine the observed effects of that antibody over time. C57BL / 6 male mice were administered one intraperitoneal injection of the antibody at 10 mg / kg (150 μl / mouse) with five mice per group. Groups were five mice sacrificed at 24 hours, 72 hours, one week, and two weeks. Analyses were the same as in Example 11, but also included hematocrit (HCT), mean corpuscular hemoglobin (MCH), and red blood cell distribution width (RDW), a measure of cell diameter related to red blood cell volume.

[0370] Administration of 10 mg / kg antibody maintained elevated hepcidin levels for 2 weeks, with reductions in serum iron and TSAT over that period. MCV levels declined due to consistent iron restriction. (Figure 10)

[0371] At 3 mg / kg, the effect of antibody on hepcidin elevation was lost by 2 weeks for the lower dose. Despite this, serum iron and TSAT remained reduced over 2 weeks, and MCV was also reduced similarly to the 10 mg / kg dose (Figure 11). Example 13

[0372] 24-hour evaluation of two IgG / kappa and two IgG / lambda anti-MTP-2 antibodies in reducing serum iron and transferrin saturation NORI-008 in a mouse IgG1 (moIgG1) 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 were included as negative control antibodies in the moIgG1 and huIgG4PE formats, respectively. The latter control antibody was a cross-reactive binder to MTP-2 but did not inhibit the enzymatic activity of either the human or mouse ECD protein in vitro.

[0373] Nine-week-old C57BL / 6 male mice were administered one intraperitoneal injection of antibody at 10 mg / kg (150 μl / mouse) with 3–5 mice per group. Mice were sacrificed 24 h after injection. Real-time PCR for hamp, Id1, 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 from all mice was determined, and transferrin saturation was calculated.

[0374] Here, we found that NORI-008, which has a very strong affinity for human and mouse MTP-2, is also active in vivo. Compared with NORI-010, it was found to be active in increasing hepcidin mRNA expression and reducing serum iron content at 24 hours (Figure 12).

[0375] 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 was successful in generating antibodies that can successfully block MTP-2 in vivo and induce a biological response. Example 14

[0376] Time course evaluation of NORI-008 after single ip injection at 10, 3, and 1 mg / kg in normal mice This experiment demonstrates the dose-time relationship of changes in serum iron concentration after ip injection of the anti-MTP-2 antibody NORI-008.

[0377] Male C57BL6J mice weighing 22–28 g, n = 5 per group, were administered a single ip injection of 2, 20, or 200 μg of the fully human IgG4 anti-MTP-2 antibody NORI-008. One group received a 200 μg dose of a human IgG4 isotype control antibody and was selected on day 1. The remaining nine groups consisted of three groups per dose of NORI-008 (2, 20, or 200 μg), with each group selected at a different dose. Blood from one group of animals treated with each dose was collected on days 1, 7, and 14 to determine serum iron levels and assess the efficacy of NORI-008.

[0378] Samples from all doses and time points were then analyzed for hepcidin expression in the liver and antibody levels in serum on day 7 (PK analysis). At 24 hours after the 20 μg dose, only 3 of 5 mice had measurable levels.

[0379] At the 200 μg dose, the NORI-008 effect lasted for at least one week in vivo, after which hepcidin expression and serum iron returned to normal at two weeks. At the 20 μg dose, the effect lasted for at least one day, after which hepcidin expression and serum iron returned to normal at one week. At the 2 μg dose, no effect was observed. The duration of effect correlates with the PK value of the antibody concentration. That is, when the antibody falls below a critical concentration, the effect on MTP-2 inhibition is lost, and hepcidin expression and serum iron return to normal. Figure 13. Example 15

[0380] In vivo evaluation of six IgG4 / kappa anti-MTP2 antibodies in normal mice at one week for reducing serum iron and transferrin saturation This study evaluated six fully human IgG4 anti-MTP-2 antibodies for their ability to reduce serum iron concentrations after ip injection.

[0381] Male C57BL6J mice weighing 25–32 g, n = 3–5 per group, were administered a single ip injection of 10 mg / kg of fully human IgG4 anti-MTP-2 antibodies. Eight groups were included in the study, and each animal received a single ip dose of 10 mg / kg of one of seven fully human IgG4PE 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 served as a positive control and benchmark anti-MTP-2 antibody for comparison with the remaining antibodies used in this experiment. All groups contained five animals, except for NORI-005 (n = 3) and NORI-002 (n = 4). Animal blood was collected on day 7 to determine serum iron concentrations and to assess iron parameters, hamp mRNA, and IgG levels of anti-MTP-2 antibodies.

[0382] All antibodies tested showed an increase in hepcidin mRNA expression and a corresponding reduction in serum iron levels. Samples from all tested groups were then analyzed for antibody levels in serum on day 7 (PK analysis), demonstrating the various levels of antibody exposure at this time point and dose. (Figure 14). Example 16

[0383] In vivo evaluation of two IgG / kappa and one IgG / lambda anti-MTP2 antibodies (10 mg / kg IP dose) in both huIgG4 and moIgG1 backbones for reducing serum iron at 1 week This study evaluated three anti-MTP-2 antibodies in fully human IgG4 or murine IgG1 configuration for their ability to reduce serum iron concentrations after subcutaneous injection.

[0384] Male C57BL6J mice weighing 25-32 g, n = 5 per group, were administered a single sc injection of 10 mg / kg of fully human IgG4 or moIgG1 anti-MTP-2 antibodies. Seven groups were included in the study, and one of three anti-MTP2 antibodies, NORI-011, NORI-003, or NORI-006, was used as a human IgG4, mouse IgG1, or human IgG4 isotype control antibody. Animal blood was collected on day 7 to check serum iron levels and evaluate the efficacy of the anti-MTP-2 antibodies.

[0385] Figure 15. Example 17

[0386] In vivo evaluation of NORI-010 after a single intraperitoneal (ip) or subcutaneous (sc) injection in normal mice with a readout 7 days after injection This experiment evaluated whether the fully human IgG4 anti-MTP-2 antibody NORI-010, after subcutaneous injection of 10 mg / kg, produced a reduction in serum iron concentrations comparable to that seen with a 10 mg / kg intraperitoneal dose.

[0387] Male C57BL6J mice weighing 23-29 g, n=4-5 / group, were administered a single 10 mg / kg sc or ip injection of a fully human IgG4 anti-MTP-2 antibody. Two groups received either a single 10 mg / kg ip dose or a single 10 mg / kg sc dose of NORI-010 (n=5 per group). The remaining two groups received a 10 mg / kg dose of a human IgG4 isotype control antibody, either ip or sc.

[0388] Animals were bled on day 7 to determine serum iron and IgG levels.

[0389] The results of this study show that serum antibody concentrations were comparable at 1 week for both ip and sc dosing methods. Consequently, serum iron reduction was also comparable for both dosing methods and was reduced by MTP-2 inhibition, as seen previously. Example 18

[0390] In vivo evaluation of dose / time response after a single ip injection of anti-MTP-2 antibodies NORI-008 and NORI-010 in normal rats This experiment evaluated the dose-time relationship of two fully human IgG4 anti-MTP-2 antibodies after ip injection.

[0391] Male Wistar rats weighing 260–320 g, n=3 / group, were administered a single ip injection of 3 or 10 mg / kg of the fully human IgG4 anti-MTP-2 antibodies NORI-008 and NORI-010. A hIgG4 isotype control antibody (labeled "Isotype") was administered by ip injection at 10 mg / kg as a negative control.

[0392] Animals were bled on days 1, 3, 7, 9, 14, and 21. Serum iron and serum antibody concentrations were measured at all time points.

[0393] NORI-010 produced a pharmacological effect at 10 mg / kg, reducing serum iron for up to 9 days, after which it returned to normal, whereas at 3 mg / kg, the effect lasted only 24 hours. NORI-008 produced an effect at 10 mg / kg, reducing serum iron for up to 9 days, after which it returned to normal, whereas at 3 mg / kg, the effect lasted only 72 hours. The PD of these antibodies correlated well with serum IgG levels, and the iron-reducing effect was lost as the antibody concentration decreased. (Figure 17) Example 19

[0394] In vivo evaluation of dose / time response after a single sc injection of anti-MTP-2 antibodies NORI-003, NORI-006, NORI-008, and NORI-010 in normal rats An in vivo evaluation of the dose / time response following a single sc injection of anti-MTP-2 antibody in normal rats was performed.

[0395] Male Wistar rats weighing 260–320 g, n = 5 / group, were administered a single sc injection of 10 mg / kg of fully human IgG4 anti-MTP-2 antibodies NORI-003, NORI-006, NORI-008, and NORI-010. A huIgG4 isotype control antibody (labeled "Isotype") was administered at 10 mg / kg as a negative control.

[0396] Animals were bled on days 1, 3, 7, 9, 14, and 21. Serum iron and serum antibody concentrations were measured at all time points.

[0397] NORI-008 and NORI-010 again showed similar duration of effect to Example 18, with their serum iron reducing effect lasting until day 9, after which it returned to normal. Both NORI-003 and NORI-006 showed more favorable duration of effect, with serum iron remaining suppressed for the entire 21-day period of the study.

[0398] PK analysis of this study shows important differences in Cmax among the four antibodies, with NORI-003 and NORI-006 having much higher serum antibody concentrations at day 3 compared to NORI-008 and NORI-010. As a result, these two antibodies exhibit more favorable PK profiles and remain above the critical serum antibody concentration for sustained MTP-2 inhibition for a longer period of time, thus prolonging their effectiveness in suppressing serum iron. Figure 18. Example 20

[0399] In vivo evaluation of NORI-010 in a mouse model of beta-thalassemia after a single ip injection The selected antibody, NORI-010, was evaluated in a heterozygous mouse model of beta-thalassemia intermedia (Hbbth3 / +), which has a heterozygous deletion of the b1 and b2 globin genes. Hbbth3 / + mice exhibit a complex phenotype similar to that of human beta-thalassemia intermedia, including hemoglobin levels between 7 and 9 g / dL, abnormal red blood cell morphology, increased reticulocyte counts, ineffective and extramedullary erythropoiesis, hepatosplenomegaly, and hepatic and splenic iron overload, all of which worsen with age. In accordance with the activity of NORI-010 observed in Examples 18-20, the antibody was injected intravenously at 10 mg / kg, and animals were sacrificed 2 weeks after this single dose (n=3 per time point). In addition, one group of animals was analyzed for hepcidin mRNA 24 hours later. A human IgG4 isotype control antibody was used at the same dose as a negative control.

[0400] The results for the following parameters were recorded: - Hepcidin mRNA levels in the liver (24 hours and 2 weeks according to the method of Example 10) - Id1 mRNA levels in the liver (24 hours and 2 weeks according to the method of Example 10) - Serum iron concentration (2 weeks according to the method of Example 10) - Calculated transferrin saturation (2 weeks according to the method of Example 10) - Mean corpuscular volume (2 weeks)

[0401] The results show that with NORI-010 administration, hepcidin levels were already elevated compared to the isotype control at 24 hours. This difference was maintained over a 2-week period. Consistent with this, serum iron levels were reduced by approximately 52% compared to the isotype control, and calculated transferrin saturation was reduced by 47% at 2 weeks compared to animals treated with the isotype control. (Figure 19) Example 21

[0402] Eight-week repeat-dose evaluation of treatment with NORI-011-M in a mouse model of beta-thalassemia with and without concomitant treatment with erythropoietin The purpose of this study was to examine the effects on hematological parameters after a longer treatment period than in Example 20. To this end, Hbbth3 / + mice (n=5 / group) were intraperitoneally injected with 10 mg / kg of NORI-011-M antibody once a week for 8 weeks. For this longer-term repeated-dose experiment, the isotype of NORI-011 was reconstituted with mouse constant regions (mouse IgG1 and mouse lambda constant regions) to avoid or suppress immunogenicity and the generation of anti-MTP2 antibodies in mice. In conjunction with this reconstitution, slight modifications were also made to the C-terminal sequence of the NORI-011 VH domain, and the engineered antibody was redesignated NORI-011-M. The sequence of NORI-011-M is shown in Table S. The control used here was mouse isotype IgG1.

[0403] In addition, this study sought to investigate the effects of coadministration of erythropoietin (EPO), which is known to improve some important parameters but has adverse effects on spleen size due to iron loading and excessive cell death. The favorable effects of this combination were confirmed in experiments in Hbbth3 / + mice in which EPO was delivered by recombinant fibroblasts overexpressing EPO and matriptase-2 activity was reduced by Tmprss6 antisense oligonucleotide treatment (Paper #164, 60th Annual Meeting of the American Society of Hematology 2018). In this study, clinical-grade EPO (darbepoetin alfa) was coadministered with NORI-011-M at 30 μg / kg once weekly to one group of the study. As a control, EPO was also coadministered with an isotype control antibody to demonstrate the effect of EPO alone on the study's readouts. Figure 20 shows the results for individual hematological parameters and hepcidin mRNA levels in liver samples.

[0404] The results again show that hepcidin levels were consistently maintained at elevated levels over the course of the 8-week experiment (a). Therefore, as seen in the previous examples, this is expected to result in iron restriction and subsequent reductions in serum iron, liver iron content (b), and MCV (f). Erythrocyte count and hemoglobin were not substantially elevated with NORI-011-M alone. However, erythrocyte maturation improved in the spleen, with the proportion of stage V (mature) cells significantly greater than in any other group (i). Increases in spleen weight were also suppressed by approximately half with NORI-011-M alone, indicating that the restriction of iron supply caused by the NORI-011-M blocker has a normalizing effect on erythrocyte maturation (k).

[0405] The use of EPO (in combination with an isotype control) had a favorable effect on red blood cell count (c) and hemoglobin (d), as expected for a stimulator of erythropoiesis. However, as previously known, increased erythropoiesis with EPO, in the context of beta-globin synthesis deficiency, led to increased cell death and an associated increase in spleen size (k), without improving maturation into functional, high-quality red blood cells (i). As expected, EPO failed to increase hepcidin levels and therefore had no effect on liver iron levels (c).

[0406] Combination treatment with NORI-011-M and EPO resulted in a favorable combination of effects. This was manifested by a more balanced overall therapeutic effect compared with EPO alone, including a sustained reduction in hepatic iron overload (b) and a reduction in splenomegaly (k), despite slightly reduced effects on red blood cell count (c) and hemoglobin (d) compared with EPO alone. Optimizing the treatment ratio between matriptase inhibition and EPO stimulation may allow for optimization of these synergistic effects in clinical practice. Example 22

[0407] Eight-week repeat-dose evaluation of treatment with NORI-011-M in a mouse model of beta-thalassemia with and without co-treatment with ActRIIB-Fc fusion protein This study demonstrates that simultaneous treatment with NORI-11-M and activin receptor IIB Fc fusion protein can achieve beneficial therapeutic effects compared with treatment with NORI-11-M alone and ActRIIB-Fc alone. This synergistic effect is due to the increased activity of ActRIIB. This may reflect the different mechanisms of action of these two agents, in that -Fc promotes the maturation of erythroid precursors during erythropoiesis, whereas NORI-11-M causes iron limitation and normalization, and therefore a slowing of erythropoiesis leading to more efficient production of more mature erythrocytes.

[0408] Work in this study was carried out over an 8-week period with multiple doses of both agents, using the methods and procedures outlined above in Example 21. As before, Hbbth3 / + mice (n=5 / group) were injected intraperitoneally with 10 mg / kg NORI-011-M antibody once per week for 8 weeks. ActRIIB-Fc was injected intraperitoneally with 10 mg / kg twice per week for 8 weeks. This doubling of ActRIIb-Fc dosing compared with antibody mirrored previously published dosing protocols for similar activin receptor ligand trap molecules (Suragani RN, Cawley SM, Li R, et al., Modified activin receptor IIB ligand trap mitigates ineffective erythropoiesis and disease complications in murine beta-thalassemia, Blood, 2014;123(25):3864-3872, doi:10.1182 / blood-2013-06-511238, and Dussiot et al., 2014, for ActRIIb-Fc).

[0409] The ActRIIB-Fc construct used in this example was a modified human ActRIIB extracellular domain (uniprot - ) with the 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. Q13705, residues 26–131).

[0410] Treatment groups were compared to healthy untreated wild-type mice ("WT") and Hbbth3 / + mice treated with a mouse IgG1 control ("MoIgG1").

[0411] Figure 21 shows the results for individual hematological parameters and hepcidin mRNA levels in liver samples. Consistent with Example 21 above, the results again showed that hepcidin levels were consistently maintained at elevated levels over the course of 8 weeks using NORI-11-M (Figure 21a), demonstrating that NORI-11-M increased hepcidin expression levels, thereby causing a decrease in liver iron levels (Figure 21b), serum iron levels, and mean corpuscular volume (MCV) (Figure 21f). Treatment with ActRIIB-Fc alone did not result in an increase in hepcidin (hamp) mRNA, and co-treatment resulted in the same outcome as using NORI-11-M alone. Thus, despite the higher dosing frequency, ActRIIB-Fc was unable to directly increase hepcidin expression or correspondingly reduce liver iron levels.

[0412] Mean corpuscular hemoglobin (MCH) was reduced in mice treated with NORI-11-M, reflecting limited iron supply, but also with ActRIIB-Fc (Fig. 21h), reflecting the production of a greater number of cells (indicated by increased total red blood cell count and hemoglobin) and therefore less hemoglobin available per cell. Mean corpuscular volume (MCV) was also reduced (Fig. 21f). Normally, a low MCV indicates microcytic anemia, but in this case, iron restriction (caused by antibodies) or hemoglobin deficiency (caused by enhanced maturation of more RBCs due to ActRIIB-Fc) leads to a reduced availability per cell, as already indicated by the MCH results.

[0413] All treatment groups improved the "quality" of the red blood cells produced, as reflected in a reduction (normalization) of red blood cell distribution width (RDW) (Figure 21g). Therefore, red blood cell uniformity improved. NORI-11-M alone slightly elevated red blood cell count (Figure 21c) but not hemoglobin (Figure 21d) or hematocrit (Figure 21e). ActRIIB-Fc had a greater effect on all of these parameters. The combination did not result in any further increases over treatment with ActRIIB-Fc alone, indicating that the maturation effect on erythropoiesis was driven by ActRIIB-Fc rather than NORI-11-M. It is noteworthy, however, that NORI-11-M did not counteract the effect of ActRIIB-Fc. Red blood cell counts were restored to wild-type levels, and both hematocrit and hemoglobin levels were significantly improved by treatment with ActRIIB-Fc alone or in combination with NORI-11-M. This is important because it indicates that the iron reduction caused by NORI-11-M does not negate the beneficial effects on erythropoiesis produced by ActRIIB-Fc. Thus, overall, the benefits of both treatments can still be achieved when used in combination, both reducing iron overload and increasing red blood cell production / maturation.

[0414] Furthermore, the two agents actually provide a greater overall benefit when used together in terms of therapeutic potential than when used alone, as shown, for example, by their effect on reducing splenomegaly.

[0415] Erythrocyte maturation improved in the spleen, with the proportion of stage V (mature) cells being much greater than in any other group (Fig. 21i). The increase in spleen weight was suppressed in both treatments compared with untreated controls, indicating that even the restriction of iron supply caused by NORI-011-M had a normalizing effect on the efficiency of erythrocyte maturation (Fig. 21k).

[0416] In Example 21, we reported that the use of EPO had a favorable effect on red blood cell count (Fig. 20c) and total hemoglobin (Fig. 20d), as expected for a stimulator of erythropoiesis. However, as is already known in the medical field, the increased stimulation of erythropoiesis by the use of EPO, in the context of continued beta-globin synthesis deficiency, also leads to increased cell death and apoptosis, and an associated further increase in spleen size and weight, known as splenomegaly (Fig. 20k). In this regard, in this example, neither the use of NORI-11-M nor the use of ActRIIB-Fc caused an increase in spleen weight compared to untreated animals (Fig. 21k). Furthermore, there was indeed a significant reduction in spleen weight, although not to the level of healthy animals. Combination treatment with NORI-11-M and ActRIIB-Fc appeared to be significantly superior to monotherapy alone by further reducing spleen weight.

[0417] array Table S below shows the sequences of the antigens, antibodies and other substances described herein. All NORI VH domains, NORI VL domains, NORI CDRs, NORI heavy chains and NORI light chains, antibodies comprising them, and their encoding nucleic acids are embodiments of the present invention.

[0418] [Table 6-1] Table 6-2 Table 6-3 Table 6-4 Table 6-5 Table 6-6 Table 6-7 Table 6-8 Table 6-9 Table 6-10 Table 6-11 Table 6-12 Table 6-13 Table 6-14 Table 6-15 Table 6-16 Table 6-17 Table 6-18 Table 6-19 Table 6-20 Table 6-21 Table 6-22 Table 6-23 Table 6-24 Table 6-25 Table 6-26 Table 6-27 Table 6-28 Table 6-29 Table 6-30 Table 6-31 Table 6-32 Table 6-33 Table 6-34 Table 6-35 Table 6-36 Table 6-37 Table 6-38 Table 6-39 Table 6-40 Table 6-41 Table 6-42 Table 6-43 Table 6-44 Table 6-45 Table 6-46 Table 6-47 Table 6-48 Table 6-49 Table 6-50 Table 6-51 Table 6-52 Table 6-53 Table 6-54 Table 6-55 Table 6-56 Table 6-57 Table 6-58 Table 6-59 Table 6-60 Table 6-61 Table 6-62 Table 6-63 Table 6-64 Table 6-65 Table 6-66 Table 6-67 Table 6-68 Table 6-69 Table 6-70 Table 6-71 Table 6-72 Table 6-73 Table 6-74 Table 6-75 Table 6-76 Table 6-77 Table 6-78 Table 6-79 Table 6-80 Table 6-81 Table 6-82 Table 6-83 Table 6-84 Table 6-85 Table 6-86 Table 6-87 Table 6-88 Table 6-89 Table 6-90 Table 6-91 Table 6-92 Table 6-93 Table 6-94 Table 6-95 Table 6-96 Table 6-97 Table 6-98 Table 6-99 Table 6-100 Table 6-101 Table 6-102 Table 6-103 Table 6-104 Table 6-105 Table 6-106 Table 6-107 Table 6-108 Table 6-109 Table 6-110

Claims

1. An isolated binder polypeptide that binds to human MTP-2 and inhibits its enzymatic activity.

2. 2. The binder polypeptide of claim 1, which binds to the serine protease catalytic domain of MTP-2.

3. 3. The binder polypeptide of claim 1 or claim 2, comprising an immunoglobulin domain in which the binding site for MTP-2 is formed by a loop region of the immunoglobulin domain.

4. A binder polypeptide according to any one of claims 1 to 3 which is an antibody, optionally a human antibody.

5. The binder polypeptide according to any one of claims 1 to 4, which binds to human MTP-2 and mouse MTP-2 and inhibits their enzymatic activity.

6. 6. The binder polypeptide of any one of claims 1 to 5, which binds to human MTP-2 containing a sequence polymorphism in which residue 253 is K or E and residue 736 is V or A.

7. 7. A binder polypeptide according to any one of claims 1 to 6, which does not bind to MTP-1 and optionally does not bind to other members of the type II transmembrane serine protease family.

8. 8. The binder polypeptide of any one of claims 1 to 7, which exhibits dose-dependent inhibition of MTP-2 serine protease activity in an enzymatic assay using MTP-2 extracellular domain and a fluorescent MTP-2 substrate at a final concentration of 50 μM.

9. 9. The binder polypeptide of claim 8, which has an IC50 of less than 100 nM in an enzymatic assay against human MTP-2 extracellular domain and a fluorescent MTP-substrate at a final concentration of 50 μM, and / or has an IC50 of less than 100 nM in an enzymatic assay against mouse MTP-2 extracellular domain and a fluorescent MTP-substrate at a final concentration of 50 μM.

10. 10. The binder polypeptide of claim 9, which has an IC50 in an enzyme assay using mouse MTP-2 extracellular domain that differs by less than 100-fold from its IC50 in said assay using human MTP-2 extracellular domain.

11. 11. The binder polypeptide of any one of claims 1 to 10, which exhibits dose-dependent inhibition of MTP-2 serine protease activity in an enzymatic assay using human MTP-2 expressed on the surface of HEK293 cells and a fluorescent MTP-2 substrate at a final concentration of 50 μM.

12. 12. The binder polypeptide of claim 11, which has an IC50 of less than 100 nM in an enzyme assay using human MTP-2 expressed on the surface of HEK293 cells and a fluorescent MTP-2 substrate at a final concentration of 50 μM.

13. competes for binding to human and / or mouse MTP-2 with an IgG comprising the VH and VL domains of any of NORI-001 to NORI-033; The NORI-001 VH domain is SEQ ID NO: 35 and the VL domain is SEQ ID NO:

44. be, The NORI-002 VH domain is SEQ ID NO: 54 and the VL domain is SEQ ID NO: 63; The NORI-003 VH domain is SEQ ID NO: 67 and the VL domain is SEQ ID NO: 63; The NORI-004 VH domain is SEQ ID NO: 74 and the VL domain is SEQ ID NO: 63; The NORI-005 VH domain is SEQ ID NO: 83 and the VL domain is SEQ ID NO: 90; The NORI-006 VH domain is SEQ ID NO: 94 and the VL domain is SEQ ID NO: 100; The NORI-007 VH domain is SEQ ID NO: 107 and the VL domain is SEQ ID NO: 114; The NORI-008 VH domain is SEQ ID NO: 125 and the VL domain is SEQ ID NO: 132; The NORI-009 VH domain is SEQ ID NO: 142 and the VL domain is SEQ ID NO: 151; The NORI-010 VH domain is SEQ ID NO: 160 and the VL domain is SEQ ID NO: 166; The NORI-010 VH domain is SEQ ID NO: 170 and the VL domain is SEQ ID NO: 166; The NORI-11-M VH domain is SEQ ID NO: 176 and the VL domain is SEQ ID NO: 166; The NORI-012 VH domain is SEQ ID NO: 182 and the VL domain is SEQ ID NO: 166; The NORI-013 VH domain is SEQ ID NO: 194 and the VL domain is SEQ ID NO: 151; The NORI-014 VH domain is SEQ ID NO: 203 and the VL domain is SEQ ID NO: 151; The NORI-015 VH domain is SEQ ID NO: 194 and the VL domain is SEQ ID NO: 216; The NORI-016 VH domain is SEQ ID NO: 203 and the VL domain is SEQ ID NO: 216; The NORI-017 VH domain is SEQ ID NO: 231 and the VL domain is SEQ ID NO: 240; The NORI-018 VH domain is SEQ ID NO: 250 and the VL domain is SEQ ID NO: 259; The NORI-019 VH domain is SEQ ID NO: 267 and the VL domain is SEQ ID NO: 273; The NORI-020 VH domain is SEQ ID NO: 283 and the VL domain is SEQ ID NO: 292; The NORI-021 VH domain is SEQ ID NO: 302 and the VL domain is SEQ ID NO: 311; The NORI-022 VH domain is SEQ ID NO: 321 and the VL domain is SEQ ID NO: 330; The NORI-023 VH domain is SEQ ID NO: 339 and the VL domain is SEQ ID NO: 346; The NORI-024 VH domain is SEQ ID NO: 356 and the VL domain is SEQ ID NO: 365; The NORI-025 VH domain is SEQ ID NO: 372 and the VL domain is SEQ ID NO: 37 7, The NORI-026 VH domain is SEQ ID NO: 387 and the VL domain is SEQ ID NO: 396; The NORI-027 VH domain is SEQ ID NO:406 and the VL domain is SEQ ID NO:412; The NORI-028 VH domain is SEQ ID NO: 422 and the VL domain is SEQ ID NO: 427; The NORI-029 VH domain is SEQ ID NO:437 and the VL domain is SEQ ID NO:444; The NORI-030 VH domain is SEQ ID NO:453 and the VL domain is SEQ ID NO:462; The NORI-031 VH domain is SEQ ID NO:472 and the VL domain is SEQ ID NO:481; The NORI-032 VH Domain is SEQ ID NO:491 and the VL Domain is SEQ ID NO:497; The NORI-033 VH domain is SEQ ID NO:507 and the VL domain is SEQ ID NO:513 The binder polypeptide according to any one of claims 1 to 12, defined as:

14. 14. The binder polypeptide of claim 13, which competes with an IgG comprising the VH and VL domains of NORI-003, NORI-006, NORI-008, or NORI-011 for binding to the serine protease catalytic domain of human and / or mouse MTP-2.

15. 15. A binder polypeptide according to any one of claims 1 to 14, which competes with aprotinin for binding to the serine protease catalytic domain of human and / or mouse MTP-2.

16. 16. The binder polypeptide of claim 15, having an IC50 of less than 100 nM, less than 50 nM, or less than 20 mM in a competition assay with labeled aprotinin for binding to human and / or mouse MTP-2.

17. 17. The binder polypeptide of any one of claims 1 to 16, having an affinity (Kd) for human MTP-2 of less than 50 nM as determined by surface plasmon resonance, and / or having an affinity (Kd) for mouse MTP-2 of less than 50 nM as determined by surface plasmon resonance.

18. 18. The binder polypeptide of claim 17, wherein the Kd for mouse MTP-2 is within 50-fold of the Kd for human MTP-2.

19. 19. The binder polypeptide of any one of claims 1 to 18, comprising an antibody heavy chain variable (VH) domain obtained by recombination of a set of germline vdj gene segments set forth in Table G for any of NORI-001 to NORI-033, and / or an antibody light chain variable (VL) domain obtained by recombination of a set of germline vj gene segments set forth in Table G for any of NORI-001 to NORI-033.

20. The VH and VL domains are NORI-001, NORI-002, NORI-003, NORI-004, NORI-005, NORI-006, NORI-007, NORI-008, NORI-009, NORI-010, and NORI-011, respectively.

20. The binder polypeptide of claim 19, obtained by recombination of a set of germline gene segments set forth in Table G for 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.

21. Germline vdj gene segments: IGHV3-9 ＊ 01, IGHD4-17 ＊ 01, and IGHJ6 ＊ 02. IGHV4-61 ＊ 01, IGHD3-22 ＊ 01, and IGHJ5 ＊ 02. IGHV3-49 ＊ 05, IGHD3-9 ＊ 01, and IGHJ4 ＊ 02, or IGHV3-13 ＊ 01, IGHD3-10 ＊ 01, and IGHJ3 ＊ 02 21. The binder polypeptide of any one of claims 1 to 20, comprising an antibody heavy chain variable (VH) domain obtained by recombinantly

22. Germline vj gene segments: IGLV2-8 ＊ 01 and IGLJ2 ＊ 01. IGKV1D-33 ＊ 01 and IGKJ5 ＊ 01. IGKV1D-33 ＊ 01 and IGKJ4 ＊ 01, and IGKV3D-7 ＊ 01 and IGKJ1 ＊ 01 22. The binder polypeptide of any one of claims 1 to 21, comprising an antibody light chain variable (VL) domain obtained by recombinantly

23. 23. The binder polypeptide according to any one of claims 1 to 22, wherein the binder polypeptide comprises 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 set of HCDRs is a set of HCDRs from any one of NORI-001 to NORI-033, and / or the set of LCDRs is a set of LCDRs from any one of NORI-001 to NORI-033.

24. the set of HCDRs is the set of CDRs of NORI-003, and the set of LCDRs is the set of LCDRs of NORI-003; HCDR1 is SEQ ID NO: 48; HCDR2 is SEQ ID NO: 49; HCDR3 is SEQ ID NO: 50; LCDR1 is SEQ ID NO: 58; LCDR2 is SEQ ID NO: 59, and LCDR3 is SEQ ID NO: 60; 24. The binder polypeptide of claim 23.

25. the set of HCDRs is the set of CDRs of NORI-006, and the set of LCDRs is the set of LCDRs of NORI-006; HCDR1 is SEQ ID NO: 48; HCDR2 is SEQ ID NO: 49; HCDR3 is SEQ ID NO: 80; LCDR1 is SEQ ID NO: 98; LCDR2 is SEQ ID NO: 59, and LCDR3 is SEQ ID NO: 88; 24. The binder polypeptide of claim 23.

26. the set of HCDRs is the set of CDRs of NORI-011, and the set of LCDRs is the set of LCDRs of NORI-011; HCDR1 is SEQ ID NO: 155; HCDR2 is SEQ ID NO: 137; HCDR3 is SEQ ID NO: 156; LCDR1 is SEQ ID NO: 164; LCDR2 is SEQ ID NO: 147, and LCDR3 is SEQ ID NO: 148; 24. The binder polypeptide of claim 23.

27. the set of HCDRs is the set of CDRs of NORI-008, and the set of LCDRs is the set of LCDRs of NORI-008; HCDR1 is SEQ ID NO: 118; HCDR2 is SEQ ID NO: 119; HCDR3 is SEQ ID NO: 120; LCDR1 is SEQ ID NO: 128; LCDR2 is SEQ ID NO: 59, and LCDR3 is SEQ ID NO: 129; 24. The binder polypeptide of claim 23.

28. a VH domain having at least 90% amino acid sequence identity with the VH domain of any of NORI-001 to NORI-033, and / or a VL domain having at least 90% amino acid sequence identity with the VL domain of any of NORI-001 to NORI-033; The NORI-001 VH domain is SEQ ID NO: 35 and the VL domain is SEQ ID NO: 44; The NORI-002 VH domain is SEQ ID NO: 54 and the VL domain is SEQ ID NO: 63; The NORI-003 VH domain is SEQ ID NO: 67 and the VL domain is SEQ ID NO: 63; The NORI-004 VH domain is SEQ ID NO: 74 and the VL domain is SEQ ID NO: 63; The NORI-005 VH domain is SEQ ID NO: 83 and the VL domain is SEQ ID NO: 90; The NORI-006 VH domain is SEQ ID NO: 94 and the VL domain is SEQ ID NO: 100; The NORI-007 VH domain is SEQ ID NO: 107 and the VL domain is SEQ ID NO: 114; The NORI-008 VH domain is SEQ ID NO: 125 and the VL domain is SEQ ID NO: 132; The NORI-009 VH domain is SEQ ID NO: 142 and the VL domain is SEQ ID NO: 151; The NORI-010 VH domain is SEQ ID NO: 160 and the VL domain is SEQ ID NO: 166; The NORI-011 VH domain is SEQ ID NO: 170 and the VL domain is SEQ ID NO: 166; The NORI-11-M VH domain is SEQ ID NO: 176 and the VL domain is SEQ ID NO: 166; The NORI-012 VH domain is SEQ ID NO: 182 and the VL domain is SEQ ID NO: 16 6, The NORI-013 VH domain is SEQ ID NO: 194 and the VL domain is SEQ ID NO: 151; The NORI-014 VH domain is SEQ ID NO: 203 and the VL domain is SEQ ID NO: 151; The NORI-015 VH domain is SEQ ID NO: 194 and the VL domain is SEQ ID NO: 216; The NORI-016 VH domain is SEQ ID NO: 203 and the VL domain is SEQ ID NO: 216; The NORI-017 VH domain is SEQ ID NO: 231 and the VL domain is SEQ ID NO: 240; The NORI-018 VH domain is SEQ ID NO: 250 and the VL domain is SEQ ID NO: 259; The NORI-019 VH domain is SEQ ID NO: 267 and the VL domain is SEQ ID NO: 273; The NORI-020 VH domain is SEQ ID NO: 283 and the VL domain is SEQ ID NO: 292; The NORI-021 VH domain is SEQ ID NO: 302 and the VL domain is SEQ ID NO: 311; The NORI-022 VH domain is SEQ ID NO: 321 and the VL domain is SEQ ID NO: 330; The NORI-023 VH domain is SEQ ID NO: 339 and the VL domain is SEQ ID NO: 346; The NORI-024 VH domain is SEQ ID NO: 356 and the VL domain is SEQ ID NO: 365; The NORI-025 VH domain is SEQ ID NO: 372 and the VL domain is SEQ ID NO: 377; The NORI-026 VH domain is SEQ ID NO: 387 and the VL domain is SEQ ID NO: 396; The NORI-027 VH domain is SEQ ID NO:406 and the VL domain is SEQ ID NO:412; The NORI-028 VH domain is SEQ ID NO: 422 and the VL domain is SEQ ID NO: 427; The NORI-029 VH domain is SEQ ID NO:437 and the VL domain is SEQ ID NO:444; The NORI-030 VH domain is SEQ ID NO:453 and the VL domain is SEQ ID NO:462; The NORI-031 VH domain is SEQ ID NO:472 and the VL domain is SEQ ID NO:481; The NORI-032 VH Domain is SEQ ID NO:491 and the VL Domain is SEQ ID NO:497; The NORI-033 VH domain is SEQ ID NO:507 and the VL domain is SEQ ID NO:513 The binder polypeptide according to any one of claims 1 to 27, defined as:

29. a VH domain comprising the NORI-003 HCDRs, wherein HCDR1 is SEQ ID NO: 48, HCDR2 is SEQ ID NO: 49, and HCDR3 is SEQ ID NO: 50, and having at least 90% amino acid sequence identity with the VH domain of NORI-003, which is SEQ ID NO: 67; and LCDR1 is SEQ ID NO: 58, LCDR2 is SEQ ID NO: 59, and LCDR3 is SEQ ID NO: No. 60, a VL domain comprising the NORI-003 LCDR and having at least 90% amino acid sequence identity with the VL domain of NORI-003, SEQ ID NO:

63.

29. The binder polypeptide of claim 28, comprising:

30. 30. The binder polypeptide of claim 29, comprising the NORI-003 antibody VH domain which is SEQ ID NO:67 and the NORI-003 VL domain which is SEQ ID NO:

63.

31. a VH domain comprising the NORI-011 HCDRs, wherein HCDR1 is SEQ ID NO: 155, HCDR2 is SEQ ID NO: 137, and HCDR3 is SEQ ID NO: 156, and having at least 90% amino acid sequence identity with the VH domain of NORI-011, which is SEQ ID NO: 170; and A VL domain comprising the NORI-011 LCDRs, wherein LCDR1 is SEQ ID NO: 164, LCDR2 is SEQ ID NO: 147, and LCDR3 is SEQ ID NO: 148, and having at least 90% amino acid sequence identity with the VL domain of NORI-011, which is SEQ ID NO:

166.

29. The binder polypeptide of claim 28, comprising:

32. 32. The binder polypeptide of any one of claims 1 to 31, comprising an antibody constant region.

33. 33. The binder polypeptide of claim 32 which is an IgG antibody.

34. 34. The binder polypeptide of claim 33, comprising a human IgG4 PE constant region.

35. 35. The binder polypeptide of claim 34, comprising the NORI-003 antibody heavy chain which is SEQ ID NO: 69, and the NORI-003 antibody light chain which is SEQ ID NO:

65.

36. 35. The binder polypeptide of claim 34, comprising the NORI-006 antibody heavy chain which is SEQ ID NO: 96, and the NORI-006 antibody light chain which is SEQ ID NO:

100.

37. 35. The binder polypeptide of claim 34, comprising a NORI-011 antibody heavy chain that is SEQ ID NO: 172, and a NORI-011 antibody light chain that is SEQ ID NO:

168.

38. 35. The binder polypeptide of claim 34, comprising the NORI-008 antibody heavy chain of SEQ ID NO: 126 and the NORI-008 antibody light chain of SEQ ID NO:

134.

39. An isolated antibody comprising a VH domain and a VL domain according to any one of claims 19 to 31.

40. comprising a VH domain and a VL domain which are the VH and VL domains of any of NORI-001 to NORI-033; The NORI-001 VH domain is SEQ ID NO: 35 and the VL domain is SEQ ID NO: 44; The NORI-002 VH domain is SEQ ID NO: 54 and the VL domain is SEQ ID NO: 63; The NORI-003 VH domain is SEQ ID NO: 67 and the VL domain is SEQ ID NO: 63; The NORI-004 VH domain is SEQ ID NO: 74 and the VL domain is SEQ ID NO: 63; The NORI-005 VH domain is SEQ ID NO: 83 and the VL domain is SEQ ID NO: 90; The NORI-006 VH domain is SEQ ID NO: 94 and the VL domain is SEQ ID NO: 100; The NORI-007 VH domain is SEQ ID NO: 107 and the VL domain is SEQ ID NO: 114; The NORI-008 VH domain is SEQ ID NO: 125 and the VL domain is SEQ ID NO: 132; The NORI-009 VH domain is SEQ ID NO: 142 and the VL domain is SEQ ID NO: 151; The NORI-010 VH domain is SEQ ID NO: 160 and the VL domain is SEQ ID NO: 166; The NORI-011 VH domain is SEQ ID NO: 170 and the VL domain is SEQ ID NO: 166; The NORI-11-M VH domain is SEQ ID NO: 176 and the VL domain is SEQ ID NO: 166; The NORI-012 VH domain is SEQ ID NO: 182 and the VL domain is SEQ ID NO: 166; The NORI-013 VH domain is SEQ ID NO: 194 and the VL domain is SEQ ID NO: 151; The NORI-014 VH domain is SEQ ID NO: 203 and the VL domain is SEQ ID NO: 151; The NORI-015 VH domain is SEQ ID NO: 194 and the VL domain is SEQ ID NO: 216; The NORI-016 VH domain is SEQ ID NO: 203 and the VL domain is SEQ ID NO: 216; The NORI-017 VH domain is SEQ ID NO: 231 and the VL domain is SEQ ID NO: 240; The NORI-018 VH domain is SEQ ID NO: 250 and the VL domain is SEQ ID NO: 259; The NORI-019 VH domain is SEQ ID NO: 267 and the VL domain is SEQ ID NO: 273; The NORI-020 VH domain is SEQ ID NO: 283 and the VL domain is SEQ ID NO: 292; The NORI-021 VH domain is SEQ ID NO: 302 and the VL domain is SEQ ID NO: 311; The NORI-022 VH domain is SEQ ID NO: 321 and the VL domain is SEQ ID NO: 330; The NORI-023 VH domain is SEQ ID NO: 339 and the VL domain is SEQ ID NO: 346; The NORI-024 VH domain is SEQ ID NO: 356 and the VL domain is SEQ ID NO: 365; The NORI-025 VH domain is SEQ ID NO: 372 and the VL domain is SEQ ID NO: 377; The NORI-026 VH domain is SEQ ID NO: 387 and the VL domain is SEQ ID NO: 396; The NORI-027 VH domain is SEQ ID NO:406 and the VL domain is SEQ ID NO:412; The NORI-028 VH domain is SEQ ID NO: 422 and the VL domain is SEQ ID NO: 427; The NORI-029 VH domain is SEQ ID NO:437 and the VL domain is SEQ ID NO:444; The NORI-030 VH domain is SEQ ID NO:453 and the VL domain is SEQ ID NO:462; The NORI-031 VH domain is SEQ ID NO:472 and the VL domain is SEQ ID NO:481; The NORI-032 VH Domain is SEQ ID NO:491 and the VL Domain is SEQ ID NO:497; The NORI-033 VH domain is SEQ ID NO:507 and the VL domain is SEQ ID NO:513 It is defined as or comprising said VH and VL domains, wherein one or more non-germline residues in the framework regions have reverted to germline. Isolated antibodies.

41. The antibody of claim 40, comprising a VH domain of NORI-003 having sequence number 67 and a VL domain of sequence number 63, or wherein one or more non-germline residues in the framework regions have reverted to germline.

42. The antibody of claim 40, comprising a VH domain of NORI-011 having sequence number 170 and a VL domain of sequence number 166, or wherein one or more non-germline residues in the framework regions have reverted to germline.

43. An isolated antibody comprising the heavy and light chains of any of NORI-001 to NORI-033.

44. A monoclonal IgG antibody comprising a NORI-003 VH domain and a NORI-003 VL domain, optionally comprising a NORI-003 antibody heavy chain that is SEQ ID NO:69 and a NORI-003 antibody light chain that is SEQ ID NO:

65.

45. A nucleic acid encoding a binder polypeptide according to any one of claims 1 to 38 or an antibody according to any one of claims 39 to 44.

46. 46. ​​An in vitro host cell comprising the nucleic acid of claim 45.

47. A composition comprising a binder polypeptide according to any one of claims 1 to 38 or an antibody according to any one of claims 39 to 44 formulated with a pharmaceutically acceptable excipient, optionally for subcutaneous administration.

48. 46. ​​A composition comprising the nucleic acid of claim 45 for in vivo gene therapy.

49. 49. A composition according to claim 47 or claim 48 for use in the treatment of the human or animal body by therapy.

50. A drug combination comprising (i) an inhibitor of MTP-2, and (ii) an antagonist of a TGFβ superfamily ligand.

51. Claims for use in treating iron overload and normalizing erythropoiesis in a patient.

51. The combination according to claim 51.

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

53. 53. The combination of claim 51 or the method of claim 52, wherein the patient has beta thalassemia (optionally beta thalassemia major), MDS, Blackfan Diamond anemia, hemochromatosis type 1, or hemochromatosis type 3.

54. 54. The combination or method of any one of claims 50 to 53, wherein the antagonist is an activin II receptor ligand trap, optionally an activin IIB receptor Fc fusion protein.

55. 55. The combination or method of claim 54, wherein the antagonist is luspatercept.

56. 55. The combination or method of claim 54, wherein the antagonist is an activin IIA receptor Fc fusion protein and optionally sotatercept.

57. A drug combination comprising (i) an inhibitor of MTP-2, and (ii) erythropoietin.

58. 58. A combination according to claim 57 for use in the treatment of anemia associated with iron overload in a patient.

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

60. 60. The combination or method of any one of claims 50 to 59, wherein the inhibitor of MTP-2 is a binder polypeptide of any one of claims 1 to 38, or an antibody of any one of claims 39 to 44.

61. In patients, Reduces the absorption of dietary iron Treating iron overload, Increases hepcidin expression from liver cells Reduces anemia caused by iron overload, Lowering serum iron levels, and / or Decreases iron saturation in transferrin 49. A method comprising administering to a patient a composition according to claim 47 or claim 48.

62. 62. The method of claim 61, wherein the patient has beta thalassemia (e.g., beta thalassemia major or intermedia), 5q-MDS, or RARS.

63. The method further comprises administering to the patient an antagonist of a TGFβ family ligand, optionally wherein the antagonist is an activin II receptor ligand trap, and optionally: an activin IIB receptor Fc fusion protein, such as luspatercept, or activin IIA receptor Fc fusion proteins, such as sotatercept; 63. The method of claim 61 or claim 62.

64. 63. The method of claim 61 or claim 62, wherein the method further comprises administering to the patient an erythropoiesis stimulating agent, optionally wherein the erythropoiesis stimulating agent is erythropoietin.

65. 63. The method of claim 61 or claim 62, comprising administering to the patient a further therapeutic agent, for example luspatercept, to reduce iron overload.

66. A composition according to claim 47 or claim 48 for use in a method according to any one of claims 61 to 65.

67. Use of a composition according to claim 47 or claim 48 for the manufacture of a medicament for the treatment of a patient comprising a method according to any one of claims 61 to 65.

68. In patients Reduces the absorption of dietary iron Treating iron overload, Increases hepcidin expression from liver cells Reduces anemia caused by iron overload, Lowering serum iron levels, and / or Decreases iron saturation in transferrin 49. A therapeutic agent for reducing iron overload for use in a method, the method comprising administering to a patient the therapeutic agent and the composition of claim 47 or claim 48.

69. 49. A therapeutic agent comprising erythropoietin (EPO) for use in a method of stimulating erythropoiesis in a patient, the method comprising administering to the patient the therapeutic agent and a composition according to claim 47 or claim 48.

70. A therapeutic agent comprising an antagonist of a TGFβ family ligand for use in a method of promoting red blood cell maturation in a patient, the method comprising administering to the patient the therapeutic agent and a composition described in claim 47 or claim 48.

71. The method of any one of claims 63 to 65, the composition for use of claim 66, the use of the composition of claim 67, or the therapeutic agent for use of any one of claims 68 to 70, wherein the method comprises the steps of administering the therapeutic agent and the composition separately and sequentially to a patient.

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