Treatment of 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] This invention relates to a drug for reducing iron overload in patients with conditions such as beta-thalassemia and myelodysplastic syndrome (MDS). [Background technology]

[0002] Iron is essential for erythropoiesis, that is, the production of red blood cells that transport oxygen from the lungs to other tissues of 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. Hepcidin, a hepatic peptide hormone, plays a central role in matching iron absorption to the body's iron requirements. Hepcidin negatively regulates cellular iron efflux by promoting the breakdown of ferropocytin, the only known iron efflux agent. Ferropocytin is expressed in cells that are major iron storage compartments, such as macrophages, and on the basolateral side of duodenal cells. Negative regulation of ferropocytin by hepcidin therefore limits iron absorption in the duodenum and iron release from iron-storing cells such as macrophages. It is well known that activation of the BMP-SMAD signaling pathway in hepatocytes via the secretion of BMP ligands (mainly BMP6) stimulates hepcidin expression. Matryptase-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 Literature 1), thereby reducing hepcidin expression and increasing dietary iron uptake and iron release from cellular storage. MTP-2 expression is induced by BMP6, which is essential for hepcidin expression, and by excess iron (Non-Patent Literature 2). Therefore, while BMP6 stimulates hepcidin expression, BMP6 also increases the expression of negative regulators that maintain a negative feedback mechanism to prevent abnormal iron regulation (Non-Patent Literature 3). Figure 1.

[0003] Iron overload, manifesting as higher-than-normal transferrin iron saturation in the blood, is a cause of increased prevalence of many genetic disorders and other conditions, including beta-thalassemia, myelodysplastic syndrome (MDS), Blackfan-Diamond anemia, sickle cell disease, polycythemia vera, and hemochromatosis.

[0004] Beta-thalassemia is a hereditary hemoglobin disorder caused by a genetic defect in the beta-globin gene. In adults, hemoglobin is normally composed of four polypeptide (globin) chains, namely two alpha-globin subunits and two beta-globin subunits, each globin subunit having a heme group in which its central iron reversibly binds to oxygen. In beta-thalassemia, incomplete hemoglobin production leads to ineffective red blood cell production and, consequently, anemia (deficiency of oxygen-carrying red blood cells). Excessive production of erythropoietin (epo), which is upregulated in response to anemia, and / or elevated levels of erythropherone, a erythroblast hormone resulting from abnormally increased and ineffective red blood cell production, have an inhibitory effect on hepcidin, an iron regulator, increasing iron absorption from the gastrointestinal tract and iron release from internal stores, leading to iron overload. Interestingly, it is not anemia itself, but iron overload that is thought to shorten life expectancy in beta-thalassemia. Increased iron utilization increases transferrin saturation and heme production, which, along with a compensatory increase in alphaglobin expression, leads to hemimicrome formation, as well as increased reactive oxygen species stress and apoptosis of erythrocyte progenitor cells. This highly stimulated but ineffective erythrocyte production results in many mature erythrocytes failing to survive, leading to splenomegaly or enlargement of the spleen, which 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, or beta-thalassemia major, is the most severe form of congenital hemolytic anemia, characterized by the absence or extreme suppression of functional beta chain synthesis. Patients require very frequent blood transfusions ("transfusion-dependent beta-thalassemia"). Patients with beta-thalassemia intermediate, on the other hand, do not require regular blood transfusions and are genetically heterozygous ("transfusion-independent beta-thalassemia"). There is a third form of beta-thalassemia called beta-thalassemia minor, which is a mild, asymptomatic state with only moderate suppression of beta chain synthesis.

[0006] A regrettable consequence of blood 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 transferrin-unbound iron, necessitating iron chelation therapy. Iron chelators can form complexes with circulating transferrin-unbound iron, disrupting the equilibrium and releasing iron from tissues, thus preventing tissue damage. However, chelators do not reduce transferrin saturation and therefore do not prevent increased hemimicron formation and increased apoptosis. Patients remain transfusion-dependent. Furthermore, iron chelators can have side effects such as renal failure, toxic neutropenia, and diarrhea. While transfusions and iron chelation have improved the prognosis of transfusion-dependent patients, iron overload, which some patients experienced, remains an unresolved clinical need today.

[0007] Similarly, there are unresolved clinical needs in the treatment of patients diagnosed with other iron-overload anemia 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, as well as various tendencies to develop acute myeloid leukemia (AML). Several forms of MDS, including MDS with chromosomal deletion 5q- (5q-MDS) and refractory anemia with ring sideroblasts (RARS), are associated with anemia and toxic iron deposition in erythrocyte precursors. These forms of MDS are often associated with decreased hepcidin levels. Often, MDS can be managed with routine transfusions, but these transfusions can lead to secondary iron overload and shortened overall survival, such as beta-thalassemia. MDS patients are therefore usually treated with iron chelators once iron overload reaches a certain threshold.

[0008] Another iron overload disorder is hereditary hemochromatosis. This is the most common genetic disorder in Caucasians and is characterized by gene mutations that result in excessive iron absorption and accumulation due to hepcidin deficiency or insensitivity. Type 1 hemochromatosis results from mutations in the HFE gene. Type 2 hemochromatosis results from mutations in either the HJV or HAMP gene. Type 3 hemochromatosis results from mutations in the TFR2 gene. Type 4 hemochromatosis results from mutations 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, as well as diabetes. The current treatment is venotomy.

[0009] Rare forms of anemia have also been shown to benefit from iron-reducing therapy in animal models. These include Blackfan-Diamond anemia and sickle cell anemia. Iron deposition in the liver (primarily parenchymal cells) is also known 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 hepatic fibrosis and cirrhosis, non-alcoholic fatty liver disease (NAFDL), and non-alcoholic steatohepatitis (NASH). Hepatic fibrosis often progresses to cirrhosis, which involves loss of liver function and progression to liver cancer.

[0010] Novel medical treatments are needed for patients with the conditions described above. While overall survival can be improved by blood transfusions and iron chelation, these treatments do not address the underlying disease pathology and have undesirable side effects as described.

[0011] One area of ​​research involves investigating the biology of iron-overload anemia using mice with genetic beta-thalassemia as a model for the human disease. Hbbth3 / + mice exhibit features similar to the intermediate form of beta-thalassemia in humans, including a complex phenotype that worsens with age, Hb levels between 7 and 9 g / dL, abnormal erythrocyte morphology, reticulocyte count, ineffective and extramyeloid erythropoiesis, hepatosplenomegaly, and iron overload in the liver and spleen (Non-Patent Literature 4).

[0012] Deletion or reduction of Tmprss6(MTP2) expression has been shown to increase hepcidin expression and correct iron overload, splenomegaly, and anemia in Hbbth3 / + mice (Non-Patent Literature 5; Non-Patent Literature 6). In the Hbbth3 / + mouse model of beta-thalassemia, reduction of Tmprss6 gene expression by using Tmprss6 siRNA formulated with lipid nanoparticles (LNPs) has been shown to induce hepcidin and weaken tissue and serum iron levels. Furthermore, treatment of Hbbth3 / + animals with LNP-Tmprss6 siRNA improved erythrocyte survival and erythropoiesis, thus substantially reducing anemia (Non-Patent Literature 7).

[0013] Guo et al. demonstrated that Hbbth3 / + mice showed reduced formation of insoluble membrane-bound globin, decreased ROS, and reduced apoptosis, as well as reduced anemia, after treatment with antisense oligonucleotides against Tmprss6 (Guo et al., 2013). These animals also showed decreased 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 excess and improved hemoglobin levels compared to Tmprss6+ thalassemia mice.

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

[0016] Fusion proteins possessing the extracellular domain of the activin type II receptor bound to the Fc portion of human IgG1 are also in clinical study. These ligand traps act on the transforming growth factor β (TGFβ) superfamily to increase late erythropoiesis. Sotatercept is an activin type IIA receptor IgG-Fc fusion protein, and ruspatercept is an activin type IIB receptor IgG-Fc fusion protein. While these proteins have been shown to significantly reduce the need for red blood cell transfusions in iron-overload anemia, they have not yet been proven sufficient to achieve transfusion independence (Non-Patent Literature 8).

[0017] Luspatercept (Reblozyl) was approved by the FDA on November 8, 2019, for the treatment of anemia in adults with beta thalassemia who require regular blood transfusions, based primarily on data provided by the Phase III BELIEVE trial. The clinical primary endpoint of this study was the proportion of patients achieving at least a 33% reduction in transfusion burden from baseline and at least a 2-unit reduction from week 13 to week 24. Only 21.4% of patients achieved this, and only 7.6% - 10.3% of patients achieved a transfusion reduction of more than 50% within the same time frame. Therefore, there is still an ongoing medical need for the treatment of beta thalassemia.

Prior Art Documents

Non-Patent Documents

[0018]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

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

[0020] The present invention relates to binder polypeptides, such as antibodies that bind to and inhibit MTP-2. 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 further conditions described herein. Various aspects of the present invention relate to binder polypeptides, their use in the manufacture of pharmaceuticals and in methods of treating patients, methods for producing binder polypeptides, nucleic acids encoding binder polypeptides, and formulations of pharmaceuticals containing binder polypeptides.

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

[0022] The binder polypeptide according to the present invention may be an antibody (e.g., IgG) or a non-antibody molecule such as an alternative polypeptide scaffold containing an engineered binding loop. The inventors describe antibodies and other binders containing a binding loop for MTP-2 (e.g., for the MTP-2 serine protease catalytic domain) that inhibit the enzymatic activity of MTP-2.

[0023] Embodiments include 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-0 Examples of antibodies include NORI-019, NORI-020, NORI-021, NORI-022, NORI-023, NORI-024, NORI-025, NORI-026, NORI-027, NORI-028, NORI-029, NORI-030, NORI-031, NORI-032, and NORI-033 ("NORI-001 to NORI-033"). These represent a diverse range 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, example antibodies have also been shown to effectively reduce hepcidin expression in vivo, which is a crucial biological process in physiological iron overload and demonstrates the potential of inhibitory anti-MTP-2 binder polypeptides to treat hematological 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 containing a set of heavy chain complementarity-determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and a VL domain containing a set of light chain complementarity-determining regions (LCDRs) LCDR1, LCDR2, and LCDR3.

[0025] The antibody may contain HCDR1, HCDR2, and / or HCDR3, which is any of NORI-001 to NORI-033, and / or the antibody may contain LCDR1, LCDR2, or LCDR3, which is any of NORI-001 to NORI-033. For example, the antibody may contain 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 contain a set of HCDRs, which are a set of VH domain HCDRs from NORI-001 to NORI-033, and / or the antibody may contain a set of LCDRs, which are a set of VL domain LCDRs from NORI-001 to NORI-033. For example, the antibody may contain NORI-001, NORI-002, NORI-003, NORI-004, NORI-005, NORI-006, NORI-007, NORI-008, NORI-009, NORI-010, NORI-011, NORI-012, NO This may include HCDRs and LCDRs of RI-013, NORI-014, NORI-015, NORI-016, NORI-017, NORI-018, NORI-019, NORI-020, NORI-021, NORI-022, NORI-023, NORI-024, NORI-025, NORI-026, NORI-027, NORI-028, NORI-029, NORI-030, NORI-031, NORI-032, or NORI-033.

[0027] The binder polypeptide may contain a VH domain having at least 90% amino acid sequence identity with any of the VH domains from NORI-001 to NORI-033, and / or the binder polypeptide may contain a VL domain having at least 90% amino acid sequence identity with any of the VL domains from NORI-001 to NORI-033. For example, the binder polypeptide may include 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, N It may include the VH domain and VL domain 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 a variant VH and / or VL domain having 90% sequence identity with the aforementioned VH and / or VL domain.

[0028] In the first configuration, the antibody is The NORI-003 HCDR contains a VH domain having at least 90% sequence identity with the VH domain of NORI-003, and VL domain containing NORI-003 LCDR and having at least 90% sequence identity with the VL domain of NORI-003 Includes.

[0029] The antibody may contain the NORI-003 VH domain and the NORI-003 VL domain. In some cases, the antibody is IgG containing the NORI-003 heavy chain and the NORI-003 light chain.

[0030] In the second configuration, the antibody is The NORI-006 HCDR contains a VH domain having at least 90% sequence identity with the VH domain of NORI-006, and VL domain containing NORI-006 LCDR and having at least 90% sequence identity with the VL domain of NORI-006 Includes.

[0031] The antibody may contain the NORI-006 VH domain and the NORI-006 VL domain. In some cases, the antibody is IgG containing the NORI-006 heavy chain and the NORI-006 light chain.

[0032] In the third configuration, the antibody is The NORI-008 HCDR contains a VH domain having at least 90% sequence identity with the VH domain of NORI-008, and VL domain containing NORI-008 LCDR and having at least 90% sequence identity with the VL domain of NORI-008 Includes.

[0033] The antibody may contain the NORI-008 VH domain and the NORI-008 VL domain. In some cases, the antibody is IgG containing the NORI-008 heavy chain and the NORI-008 light chain.

[0034] In the fourth configuration, the antibody is The NORI-011 HCDR contains a VH domain having at least 90% sequence identity with the VH domain of NORI-011, and VL domain containing NORI-011 LCDR and having at least 90% sequence identity with the VL domain of NORI-011 Includes.

[0035] The antibody may contain the NORI-011 VH domain and the NORI-011 VL domain. In some cases, the antibody is IgG containing the NORI-011 heavy chain and the NORI-011 light chain.

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

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

[0038] Inhibition of MTP-2 enzymatic activity can be determined by an in vitro assay for inhibition of serine protease cleavage of the MTP-2 substrate to produce a detectable product. Such an in vitro enzyme assay may include the steps of 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 may be included instead). The enzyme assay can be performed with various concentrations of polypeptide to create a dose-response curve from which an IC50 value can be calculated. Thus, inhibitors according to the present invention can be identified by 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 50 μM final concentration of fluorescent MTP-2 substrate. A typical substrate is Boc-Gln-Gly-Arg-AMC, currently available as Baychem 4016429. In such an assay, MTP-2 may have an activity rate of 0.075 U / μl. MTP-2 is supplied as a purified protein in solution, e.g., the MTP-2 extracellular domain. Thus, the IC50 of the binder polypeptide is the activity rate of purified MTP-2 cells with an activity rate of 0.075 U / μl in the presence of 50 μM of the fluorescent substrate Boc-Gln-Gly-Arg-AMC. This can be determined by an enzyme assay using the extradomain. Binder polypeptides may have an IC50 of less than 100 nM in such assays.

[0040] Alternatively, inhibition may be performed in vitro using MTP-2 expressed on the cell surface in a cell-based assay, for example, using HEK293 cells.

[0041] The effects of MTP-2 inhibition (e.g., reduction in hepcidin expression, measurable as a reduction in the mRNA of its coding gene hamp) are further detected in vivo, confirming their inhibitory activity and biological association.

[0042] In various embodiments, the potency of the inhibitor is quantified according to its IC50, which is measured in an in vitro assay for inhibition of MTP-2 enzyme activity (e.g., the assay described above). Preferably, the binder polypeptide according to the present 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 enzyme 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 for reference to one or more anti-MTP-2 antibodies described herein. For example, an antibody containing 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 IgG. For example, a binder polypeptide according to the present invention may have an IC50 of 25% or 10% of the IC50 of any of NORI-001 to NORI-033, e.g., NORI-003 IgG, NORI-006 IgG, NORI-008 IgG, NORI-009 IgG, or NORI-011 IgG, or a binder polypeptide according to the present invention may have an IC50 lower than the IC50 of the reference antibody. "Within x% of" means that the IC50 of the test binder polypeptide is at least x% greater and at least x% less than the IC50 of the reference antibody.

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

[0045] In one embodiment, the assay for MTP-2 inhibition is performed using human MTP-2. In another embodiment, the assay for MTP-2 inhibition is performed using non-human (e.g., mouse, rat, or cynomolgus monkey) MTP-2. Comparison of the inhibitory efficacy of binder polypeptides in the same enzyme assay using MTP-2 from different species provides an indicator of the interspecies cross-reactivity of the binder polypeptide. Generally, interspecies cross-reactivity is desirable because it allows for the possibility of testing the binder polypeptide in vivo in multiple species. For example, preclinical studies in experimental animals (e.g., mice, rats, or cynomolgus monkeys) can be performed before clinical studies in the target species (e.g., humans). Preferably, the binder polypeptide according to the present invention is cross-reactive for binding to and inhibiting MTP-2 from multiple species. Preferably, the binder polypeptide according to the present invention binds to and inhibits human and mouse MTP-2. More preferably, the binder polypeptide according to the present invention binds to and inhibits human, mouse, rat, and cynomolgus monkey MTP-2.

[0046] The 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%, 25%, or 20% of its IC50 in an in vitro assay for inhibition of human MTP-2 enzyme activity.

[0047] The 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 less than 100-fold, less than 50-fold, less than 10-fold, less than 5-fold, or less than 2-fold different 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 to MTP-2 of one species with its affinity to MTP-2 of another species. The binding affinity (K) is determined, for example, by surface plasmon resonance. D ) are compared. K of binding polypeptides to non-human (e.g., mouse, rat, and / or cynomolgus monkey) MTP-2 D It binds its K to human MTP-2. D It differs by less than 50 times, less than 10 times, less than 5 times, or less than 2 times.

[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 optionally, other isolated fragments or domains selected according to the MTP-2 region to which the reference molecule binds. For example, a binder polypeptide according to the present invention may compete with aprotinin for binding to MTP-2. A binder polypeptide according to the present invention may compete for binding to MTP-2 with any anti-MTP-2 antibody described herein. For example, an antibody containing 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 IgG. For example, in various embodiments, the binder polypeptide is NORI-003 IgG, NORI-006 IgG, NORI-008 IgG, or NORI-011 IgG for binding to MTP-2. It may compete with IgG. Alternatively, the reference antibody is provided as an scFv. For example, in various embodiments, the binder polypeptide is NORI-003 scFv, NORI-006 scFv, NORI-008 scFv, etc., for binding to MTP-2. It may compete with scFv or NORI-011 IgG.

[0050] IC50 can be determined in a competitive assay. For example, a 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 58-amino acid aprotinin mature sequence shown in Table S can be used in the competitive assay.

[0051] The nucleic acids encoding the binder polypeptides described herein are also provided as cells containing such nucleic acids. In vitro host cells may contain nucleic acids, which may be incorporated into the host cell's cellular (e.g., genomic) DNA or transiently transfected (e.g., plasmid DNA).

[0052] These and other aspects and embodiments of the present invention, including methods for producing binder polypeptides, pharmaceutical compositions, and methods for 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 the uptake of dietary iron and the release of iron from cellular stores in the body. MTP-2 inhibitors (such as antibodies against the serine protease domain) can be used to treat iron overload, which is characteristic of diseases such as beta-thalassemia and other diseases that cause toxic accumulation of iron. Combinations of MTP-2 inhibitors with activin receptor ligand traps or erythropoietin may yield further therapeutic effects.

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

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

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

[0057] 3. Antibodies, sometimes human antibodies, and binder polypeptides as described in item 2.

[0058] 4. MTP-2 is human MTP-2, a binder polypeptide as described in any of items 1-3.

[0059] 5. MTP-2 is human MTP-2 and mouse MTP-2, the binder polypeptides described in item 4.

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

[0061] 7. A binder polypeptide described in any of items 1-6, 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. A binder polypeptide described in any one of items 1 to 7, which exhibits dose-dependent inhibition of MTP-2 serine protease activity in an enzyme assay using the MTP-2 extracellular domain and a final concentration of 50 μM of fluorescent MTP-2 substrate.

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

[0064] 10. A binder polypeptide as described in item 8 or 9, having an IC50 of less than 100 nM in an enzymatic assay against the mouse MTP-2 extracellular domain and a final concentration of 50 μM of fluorescent MTP-substrate.

[0065] 11. The binder polypeptide described in item 10, which, in an enzyme assay using the mouse MTP-2 extracellular domain, has an IC50 that is less than 100-fold different from the IC50 of the assay using the human MTP-2 extracellular domain.

[0066] 12. A binder polypeptide according to any of items 1 to 11, which 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 final concentration of 50 μM of fluorescent MTP-2 substrate.

[0067] 13. A binder polypeptide as described in 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 final concentration of 50 μM of fluorescent MTP-2 substrate.

[0068] 14. A binder polypeptide described in any of items 1-13 that competes with IgG containing the VH and VL domains of any of NORI-001 to NORI-033 for binding to human and / or mouse MTP-2.

[0069] 15. Binder polypeptides described in item 14 that compete with IgG containing 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 described in any of items 1-15 that competes with aprotinin for binding to the serine protease catalytic domain of human and / or mouse MTP-2.

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

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

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

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

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

[0076] 22. The binder polypeptide described in item 21, wherein the Kd for mouse MTP-2 is no more than 50 times that for human MTP-2.

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

[0078] 24. The VH domain and VL domain 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, The binder polypeptide according to item 23, obtained by recombination of a set of germline gene segments shown 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 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 The binder polypeptide according to any of items 1 to 24, comprising an antibody heavy chain variable (VH) domain obtained by recombination of

[0080] 26. 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 <000002​ A binder polypeptide according to any of items 1 to 25, comprising an antibody light chain variable (VL) domain obtained by recombination.

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

[0082] 28. The HCDR set is the CDR set of NORI-003, and the LCDR set is the LCDR set of NORI-003, the binder polypeptide as described in item 27.

[0083] 29. The HCDR set is the CDR set of NORI-006, and the LCDR set is the LCDR set of NORI-006, the binder polypeptide as described in item 27.

[0084] 30. The HCDR set is the CDR set of NORI-011, and the LCDR set is the LCDR set of NORI-011, the binder polypeptide as described in item 27.

[0085] 31. The HCDR set is the CDR set of NORI-008, and the LCDR set is the LCDR set of NORI-008, the binder polypeptide as described in item 27.

[0086] 32. A binder polypeptide according to any of items 1 to 31, comprising 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.

[0087] 33. A VH domain containing NORI-003 HCDR and having at least 90% amino acid sequence identity with the VH domain of NORI-003, and A VL domain containing NORI-003 LCDR and having at least 90% amino acid sequence identity with the VL domain of NORI-003. Binder polypeptides, including those listed in item 32.

[0088] 34. The binder polypeptide described in item 33, comprising the NORI-003 antibody VH domain and the NORI-003 VL domain.

[0089] 35. A VH domain containing NORI-006 HCDR and having at least 90% amino acid sequence identity with the VH domain of NORI-006, and A VL domain containing NORI-006 LCDR and having at least 90% amino acid sequence identity with the VL domain of NORI-006. Binder polypeptides, including those listed in item 32.

[0090] 36. The binder polypeptide described in item 32, comprising the NORI-011 VH domain and the NORI-011 VL domain.

[0091] 37. A VH domain containing NORI-011 HCDR and having at least 90% amino acid sequence identity with the VH domain of NORI-011, and A VL domain containing NORI-011 LCDR and having at least 90% amino acid sequence identity with the VL domain of NORI-011. Binder polypeptides, including those listed in item 36.

[0092] 38. The binder polypeptide described in item 32, comprising the NORI-008 VH domain and the NORI-008 VL domain.

[0093] 39. A VH domain containing NORI-008 HCDR and having at least 90% amino acid sequence identity with the VH domain of NORI-008, and A VL domain containing NORI-008 LCDR and having at least 90% amino acid sequence identity with the VL domain of NORI-008. Binder polypeptides, including those listed in item 38.

[0094] 38. Binder polypeptide as described in item 37, comprising the NORI-008 VH domain and the NORI-008 VL domain.

[0095] 39. A binder polypeptide described in any of items 1 to 38, including an antibody constant region.

[0096] 40. IgG antibody, the binder polypeptide described in item 39.

[0097] 41. A binder polypeptide as described in item 40, containing the human IgG4PE constant region.

[0098] 42. A binder polypeptide as described in item 41, comprising the NORI-003 antibody heavy chain and the NORI-003 antibody light chain.

[0099] 43. A binder polypeptide as described in item 41, comprising the NORI-006 antibody heavy chain and the NORI-006 antibody light chain.

[0100] 44. A binder polypeptide as described in item 41, comprising the NORI-011 antibody heavy chain and the NORI-011 antibody light chain.

[0101] 45. A binder polypeptide as described in item 41, comprising the NORI-008 antibody heavy chain and the NORI-008 antibody light chain.

[0102] 46. ​​An isolated antibody containing the VH domain and VL domain described in any of items 23-38.

[0103] 47. Includes VH domains and VL domains that are any of NORI-001 to NORI-033, Alternatively, the VH and VL domains include the VH and VL domains in which one or more non-germline residues in the framework region are returned to the germline domain. Isolated antibodies.

[0104] 48. The antibody described in item 47, comprising the VH and VL domains of NORI-003, or the VH and VL domains wherein one or more non-germline residues in the framework region are returned to germline.

[0105] 49. The antibody according to item 47, comprising the VH and VL domains of NORI-006, or the VH and VL domains wherein one or more non-germline residues in the framework region are returned to germline.

[0106] 50. The antibody according to item 47, comprising the VH and VL domains of NORI-011, or the VH and VL domains wherein one or more non-germline residues in the framework region are returned to germline.

[0107] 51. The antibody described in item 47, comprising the VH and VL domains of NORI-008, or the VH and VL domains wherein one or more non-germline residues in the framework region are returned to germline.

[0108] 52. Isolated antibodies containing the heavy and light chains 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, NORI-011 antibody heavy chain and NORI-011 antibody light chain, or NORI-008 antibody heavy chain and NORI-008 antibody light chain A monoclonal IgG antibody containing [this antibody].

[0110] 54. A nucleic acid encoding a binder polypeptide as described in any of items 1-45 or an antibody as described in item 46 or item 53.

[0111] 55. In vitro host cells containing the nucleic acids described in item 54.

[0112] 56. A composition comprising a binder polypeptide as described in any of items 1 to 45 or an antibody as described in any of items 46 to 53, formulated with pharmaceutically acceptable excipients.

[0113] 57. The composition described in item 56 for subcutaneous administration.

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

[0115] 59. For use in the treatment of the human or animal body by therapeutic means, items 56-5 The composition described in any of 8.

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

[0117] 61. The combination described in item 60 for use in the treatment of iron overload and normalization of red blood cell production in patients.

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

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

[0120] 64. The antagonist is an activin II receptor ligand trap, in any combination or method described in items 60-63.

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

[0122] 66. The antagonist is ruspatercept, in any combination or method described in item 65.

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

[0124] 68. The antagonist is a sotatercept, as described in item 67, in combination or by any other method.

[0125] 69. A combination of drugs including (i) an MTP-2 inhibitor and (ii) erythropoietin.

[0126] 70. The combination described in item 69 for use in the treatment of anemia associated with iron overload in patients.

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

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

[0129] 72. In patients, Reduces the absorption of dietary iron. Treat iron excess It increases the expression of hepcidin from liver cells. It reduces anemia caused by iron overload. Lowers serum iron levels, and / or It reduces the iron saturation of transferrin. A method comprising the step of administering to a patient a composition described in any of items 56 to 58.

[0130] 73. The patient has beta-thalassemia (e.g., beta-thalassemia major or intermedia), 5q-MDS, or RARS, as described in item 72.

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

[0132] 75. The antagonist is an activin II receptor ligand trap, as described in item 74.

[0133] 76. The antagonist is an activin IIB receptor Fc fusion protein, as described in item 75.

[0134] 77. The antagonist is a ruspatercept, as described in item 76.

[0135] 78. The antagonist is an activin IIA receptor Fc fusion protein, as described in item 75.

[0136] 79. The antagonist is a sotatercept, as described in item 76.

[0137] 80. The method according to item 72 or item 73, further comprising the step of administering a erythropoiesis-stimulating agent to a patient, wherein the erythropoiesis-stimulating agent is optionally erythropoietin.

[0138] 81. The method according to item 72 or item 73, further comprising the step of administering to the patient an additional therapeutic agent, such as ruspatercept, in order to reduce iron excess.

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

[0140] 83. Use of any of the compositions described in any of items 56 to 58 for the manufacture of a drug for the treatment of a patient, which includes the method described in any of items 72 to 81.

[0141] 84. In patients Reduces the absorption of dietary iron. Treat iron excess It increases the expression of hepcidin from liver cells. It reduces anemia caused by iron overload. Lowers serum iron levels, and / or It reduces the iron saturation of transferrin. A therapeutic agent for reducing iron excess for use in a method, the method comprising the step of administering the therapeutic agent and a composition described in any of items 56 to 58 to a patient.

[0142] 85. A therapeutic agent comprising erythropoietin (epo) for use in a method for stimulating red blood cell production in a patient, the method comprising the step of administering the therapeutic agent and a composition described in any of items 56 to 58 to a patient.

[0143] 86. TGFβ for use in methods to promote erythrocyte maturation in patients A therapeutic agent comprising a milly ligand antagonist, the method comprising the step of administering the therapeutic agent and a composition described in any of items 56 to 58 to a patient.

[0144] 87. A method comprising the step of administering a therapeutic agent and composition to a patient individually and sequentially, according to any of items 74 to 81, a composition for use as described in item 82, a composition for use as described in item 83, or a therapeutic agent for use as described in any of items 84 to 86.

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

[0146] Isoform 1, the standard isoform of MTP-2, is a protein with a molecular weight of 90 kDa and consisting of 811 amino acids. It has a conserved structure similar to closely related TTSP family members such as matryptase-1 and enteropeptidase, and its extracellular structure consists of a main domain with a small N-terminal intracellular signal peptide that functions as a signal anchor in a single transmembrane domain, followed by sea urchin sperm protein, enteropeptidase, and agrin (SEA) domains, two complement factor C1r / C1s, sea urchin embryo growth factor, and bone morphogenetic protein (CUB) domains, and three low-density lipoprotein receptor (LDLR) class A repeats, and a C-terminal serine protease (SP) domain. The SP domain contains a highly conserved catalytic amino acid triplet necessary for enzymatic function: histidine (617), aspartic acid (668), and serine (762). Figure 2.

[0147] Like other TTSP members, the precise 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 further endogenous proteins play crucial roles. MTP-2 is synthesized within the endoplasmic reticulum membrane and transported to the cell surface as an inactive enzymatic precursor, where it is autocleaved at an arginine residue within a highly conserved activation motif between the CUB2 domain and the serine protease. The MTP-2 serine protease domain has four essential disulfide bonds, one of which, importantly, attaches the domain to the membrane-bound mainstem.

[0148] The cleaved form of MTP-2 exhibits an active form, largely remaining membrane-bound, in which case MTP-2 can cleave other membrane-bound targets on the cell surface. However, in vitro overexpression of MTP-2 has revealed a "fluxed" active form of MTP-2, which is observed in the supernatant of cultured cells. It remains unclear whether this fluxed form is part of the innate MTP-2 biology with in vivo function, or simply a result of overexpression in cell-based systems.

[0149] The amino acid sequence of MTP-2 is shown in Table S. For example, human MTP-2 is Uniprot It has the amino acid sequence ID Q8IU80 and includes an N-terminal leader sequence and a cytoplasmic domain, a transmembrane domain, and an extracellular domain (ECD). The ECD contains amino acids 84-811 of the full-length protein. The MTP-2 fragment containing the ECD, amino acids 78-811, can be produced by recombination and used in the assays described herein (e.g., in histagged form). The production of MTP-2 for use in the assay is detailed in Example 5.

[0150] The binder polypeptide according to the present invention is MTP-2 expressed on the cell surface, isolated and It can bind to one, more, or all of the MTP-2 ECD and elution-type soluble MTP-2 ECD, and inhibit them.

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

[0152] Because MTP-2 processing is complex, involving a large portion of the protein structure, mutations in the protein can lead to loss of function. In addition, TMPRSS6 polymorphisms such as rs855791 are known, which can result in increased MTP-2 activity and more efficient inhibition of hepcidin. The R576A mutation alters a critical arginine residue necessary for SP domain cleavage and complete MTP-2 activation, thus maintaining the protein as an inactive enzyme precursor. The S762A mutation alters a critical serine residue within the catalytic triplet of the SP domain, completely repressing its activity. Thus, since self-cleavage requires self-activation, the protein also remains an inactive enzyme precursor. The E114K mutant has been described in patients with non-functional MTP-2 expression. This mutation is located within the SEA domain and is therefore highly likely to interfere with the proper delivery of the protein to the cell surface.

[0153] The following four variants of MTP-2 are known, covering approximately 92% of the human population: Isoform 1 (27.4%); rs855791 SNP (27.2%) causes a single amino acid change from valine to alanine at position 736; Variants possessing both V736A and further K253E mutations (25.9%); and A variant of K253E alone (11.4%).

[0154] The binder polypeptide preferably binds to all four such variants and is therefore suitable for treating all or most of the human population by inhibiting the MTP-2 variant expressed by the majority of the human population. Thus, the binder polypeptide can bind to human MTP-2 including the 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, has a full length of 811 amino acids and is mainly expressed in the testes. Isoform 2 is the major isoform in the liver and lacks nine intracellular amino acids at the N-terminus (802 amino acids). This N-terminal region is thought to be involved in the internalization of membrane-bound MTP-2 and therefore internalizes more slowly than isoform 1. Isoform 3 is also mainly expressed in the testes, along with isoform 1. It has nine N-terminal amino acids but lacks the SP domain and therefore utilizes an alternative splicing variant of exon 10 to promote the expression of a truncated form that is functionally inactive. Isoform 4 has the same exon as isoform 2 but also has an additional exon of 22 amino acids between exons 16 and 17 that disrupts the function of the SP domain and is similarly functionally inactive. Isoform 4 is thought to be expressed in tissues that also express isoform 2, and since isoforms 3 and 4 have lost their function, they are considered to be dominant-negative regulators of isoforms 1 and 2. Expression of isoform 3 or 4 has been shown to block isoform 2-mediated HJV cleavage.

[0156] The binder polypeptide may bind to at least the active isoforms 1 and 2. The binder polypeptide may optionally bind to isoform 3. The binder polypeptide may optionally bind to isoform 4. The binder polypeptide may not bind to isoform 3. The binder polypeptide may not bind to isoform 4. The absence of binding to inactive isoforms may be advantageous for therapeutic molecules intended to inhibit MTP-2 activity. The lack of binding to the serine protease catalytic domains of isoform 1 and / or isoform 2, as well as the lack of binding to isoforms 3 and / or isoform 4, may be advantageous.

[0157] Integration with MTP-2 As described above, MTP-2 is a multi-domain protein, and various mutations in this protein are known to lead to 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 can bind to the serine protease catalytic domain of MTP-2. The binder polypeptide can bind to self-activated MTP-2. The binder polypeptide can bind to the MTP-2 ECD. The binder polypeptide can bind to the MTP-2 enzyme precursor.

[0159] Binder polypeptides that bind to the serine protease catalytic domain are identified as binding to MTP-2 containing the domain but not to MTP-2 lacking the domain. A "headless" variant of MTP-2 can be constructed, having a C-terminal truncation that deletes the serine protease domain but still containing the remaining ECD. The absence of binding to the full-length MTP-2 ECD and 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 an HTRF assay or by surface plasmon resonance, for which an example protocol is 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, but do not exhibit dose-dependent binding to headless MTP-2 ECD in the same assay.

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

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

[0162] Competition between binder polypeptides can also be determined. For example, a binder polypeptide may compete with any of NORI-001 to NORI-033 antibodies containing VH and VL domains (e.g., IgG or scFv), or with IgG containing full heavy and light chains. A binder polypeptide may compete with, for example, NORI-003 scFv. A binder polypeptide may compete with NORI-006 scFv. A binder polypeptide may compete with NORI-011 scFv. A binder polypeptide may compete with NORI-008 scFv.

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

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

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

[0166] Materials and methods for HTRF competitive assays The antibody was titrated at a starting concentration of 200 nM in DPBS (Gibco-14190144) containing HTRF buffer (0.1% BSA (Sigma-A7906) and 0.53 M potassium fluoride (Sigma-60240-250G)) at a 4-fold concentration. 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 a 4-fold final concentration in HTRF buffer (huMTP-2 = 40 nM, and moMTP-2 = 240 nM) and seeded at 5 μL / w. The moIgG1-based anti-MTP-2 mAb (NORI-037) was then diluted to a 4-fold final concentration of 1.2 nM using 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 4-fold final concentration of 20 nM in HTRF buffer and seeded at 5 μL / w. The plates were incubated in RT, dark for at least 3 hours. The plates were read at 1, 2, and 3 hours using the HTRF 100 flash protocol with an EnVision plate reader (Ex: 340 nm, Em1: 620 nm, Em2: 665 nm).

[0167] Bond affinity The binder polypeptide has an affinity (K) for human MTP-2, with concentrations of less than 100 nM, less than 50 nM, less than 25 nM, or less than 10 nM. D The binder polypeptide may have affinity (Kd) to 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 affinity (Kd) to 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 affinity (Kd) to cynomolgus monkey 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 binding of human MTP-2 is K DIt may be less than 5 nM, for example, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, or less than 0.1 nM. D This is, in some cases, at least 0.001 nM, for example, at least 0.005 nM.

[0169] The binder polypeptide is, for example, within the affinity range shown in Table K or Table W in Example 6 (K D ) may indicate the binding of the binder polypeptide K to MTP-2. D This refers to the K of one of NORI-001 to NORI-033 IgG or scFv (e.g., NORI-009 IgG). D It is the same as or lower than K. D This is the K of aprotinin D It is the same as or lower than that.

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

[0171] In short, SPR can be performed at 25°C by capturing the binder polypeptide onto a chip for 60 seconds at a concentration of 1 μg / ml at 10 μl / min (capturing approximately 35–50 RU), injecting MTP-2 (analyte) for 120 seconds (association time) at 30 μl / min, and monitoring dissociation for 600 seconds. The analyte may be injected at concentrations of 100, 25, 6.25, 1.56, and 0 nM. A sensorgram of the binder polypeptide is created, and the data is fitted to a 1:1 interaction model (e.g., using Biacore evaluation software with overall fitted Rmax, ka, kd, and RI=0).

[0172] Affinity quantification can be performed using SPR with a monovalent antigen-binding polypeptide arm, e.g., an antibody Fab or Fv containing an antigen-binding site, or a heterodimer immunoglobulin (e.g., IgG) having a single antigen-binding arm for the antigen of interest. Alternatively, it may be convenient to determine affinity to IgG containing a bivalent antigen-binding polypeptide arm, e.g., a homodimer antigen-binding arm. SPR may (directly or indirectly) contain a coating dimer of the antigen-binding polypeptide arm on a biosensor chip, the antigen-binding polypeptide arm is exposed to antigens in buffer solutions of various concentrations, binding is detected, and the equilibrium dissociation constant KD of the binding interaction is calculated. SPR may be performed at 25°C. Suitable buffer solutions are 150mM NaCl, 0.05% surfactant (e.g., P20), and 3mM EDTA, pH 7.6. HBS-P 1×(HEPES 10mM, pH 7.4, NaCl) containing 2.5mM CaCl2. A buffer (150 mM, 3 mM EDTA, 0.05% polysorbate 20, pH 7.6) is an example buffer. The combined data can be fitted to a 1:1 model using standard algorithms that may be specific to the instrument used. Various SPR instruments are known, such as Biacore®, ProteOn XPR36® (Bio-Rad®), and KinExA® (Sapidyne Instruments, Inc.).

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

[0174] Cross-reactivity Regulatory bodies may require demonstrating the therapeutic efficacy of candidate therapeutic molecules in experimental 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 derived from one or more non-human mammals. Therefore, the binder should be able to cross-react with both non-human MTP-2 and human MTP-2. P-2 can also bind.

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

[0176] Interspecies cross-reactivity binding molecules may have affinity differences 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 for binding to human and non-human antigens. In other words, the KD for binding to 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 to the extracellular domain of a non-human antigen.

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

[0178] A binding molecule can also be considered interspecies cross-reactive if the KD of the binding of both antigens meets a threshold, for example, if the KD of the binding of the human antigen and the KD of the binding of the non-human antigen are both 10 mM or less, preferably 5 mM or less, and more preferably 1 mM or less. The KD may 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 the cell, for example, using FACS. HTRF can also be used to determine the binding of multiple species of MTP-2 and their cross-reactivity.

[0180] Binder polypeptides may have some ability to block enzyme activity via fluorescence readout using one or more or all of the following MTP-2 substrates from multiple species (e.g., human, 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] While interspecies cross-reactivity for binding to antigens of different species can be advantageous, binder selectivity for MTP-2 is still necessary to avoid undesirable side effects. Therefore, in the body, MTP-2 is preferably the only antigen to which the antigen-binding site of the binder polypeptide binds. Nevertheless, binder polypeptides may be engineered to include additional binding sites, and antibodies containing an antibody constant region may optionally bind to, for example, one or more Fc receptors.

[0182] Binder polypeptides may not bind to MTP-1 (e.g., human MTP-1) in some cases. Binder polypeptides may not bind to MTP-3 (e.g., human MTP-3) in some cases. Binder polypeptides may not bind to other members of the type II transmembrane serine protease family in some cases.

[0183] MTP-2 inhibition MTP-2 is primarily expressed in hepatocytes and plays a major role in iron metabolism by regulating hepcidin expression from hepatocytes. It is now known that hepcidin expression, a key regulator of iron homeostasis, is controlled by bone morphogenetic protein (BMP) growth factor. This growth factor binds to type I and type II BMP receptors present in hepatocytes, inducing the BMP / sons of mothers against decapentaplegic (SMAD) signaling pathway. Phosphorylation of the Smad1,5,8 / Smad4 complex downstream of the BMP receptor increases the expression of the HAMP gene encoding hepcidin, thereby increasing hepcidin secretion. Hepcidin binds to the iron transporter ferroportin, present in duodenal cells, macrophages, and hepatocytes, inducing its internalization and degradation, thus reducing blood iron levels by decreasing the amount of iron entering the bloodstream. MTP-2 is thought to negatively regulate hepcidin expression by selectively cleaving members of the BMPR complex on the surface of hepatocytes, thereby silencing BMP / SMAD signaling. One proposed enzymatic target of MTP-2 is hemoduvelin (HJV), a co-receptor of the BMPR complex required for maximum BMP / SMAD signaling. However, while studies have shown that MTP-2 can cleave HJV and produce specific cleavage products, mice masked for MTP-2 show a contradictory decrease in the expression of membrane-bound HJV, while TMPRSS6 KO mice show increased levels of cleaved HJV. More recently, studies have shown that MTP-2 most likely inhibits hepcidin expression by cleaving multiple members of the BMPR complex.

[0184] Tissue-wide scans of TMPRSS6 mRNA expression also reveal low levels of MTP-2 expression in the testes, although the role of MTP-2 in this context is largely unknown. Due to localized MTP-2 expression, both TMPRSS6 KO mice and humans with loss-of-function MTP-2 mutations exhibit a phenotype with significantly elevated iron levels and no further side effects, suggesting that its further role in iron regulation is limited and therefore not a major concern with anti-MTP-2 therapy.

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

[0186] MTP-2 inhibition refers to the inhibition of the enzymatic activity of MTP-2. MTP-2 is a serine protease, and inhibitors may inhibit the catalyst of serine protease cleavage of its substrate by mature, active MTP-2, and / or inhibit the self-activation of the MTP-2 enzyme precursor by catalyst of serine protease cleavage.

[0187] In various embodiments, a binder polypeptide may bind to the serine protease catalytic domain of MTP-2. The serine protease catalytic domain contains the enzymatic active site of MTP-2. Inhibition may be a result of steric hindrance to the enzyme-substrate interaction, caused by the binder polypeptide binding to MTP-2 and partially or completely masking the enzymatic active site to reduce substrate binding. Inhibition may also be a result of the binder polypeptide reducing its enzymatic activity by inducing inactivation of a conformational change in the serine protease catalytic domain or by biasing the serine protease catalytic domain to an inactive conformation. 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 enzyme assays using purified MTP-2 ECD and a fluorescent substrate, and enzyme assays using MTP-2 expressed on the cell surface 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, for example, 50 μM. Binder polypeptides that inhibit 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 enzymatic assay against the human 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 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 enzymatic assay against a 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 may 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.

[0191] As described above, preferably, the binder polypeptide is interspecies cross-reactive and therefore inhibitory to MTP-2 of two or more species, for example, human and non-human (e.g., mouse, rat, or cynomolgus monkey) MTP-2. Parameters for quantifying cross-reactivity in assays are discussed elsewhere in this specification.

[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 final concentration of 50 μM of fluorescent MTP-2 substrate. 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 may 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.

[0193] Inhibition of MTP-2 enzymatic activity is further detectable 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 are obtained from in vivo assays. In mice administered with the binder polypeptide, inhibition of MTP-2 enzymatic activity may result in increased hamp mRNA, decreased serum iron, and decreased transferrin saturation (TSAT). To measure these effects, the binder polypeptide is administered to wild-type mice (e.g., at 10 mg / kg). Within 24 hours after administration, the binder polypeptide increases hamp mRNA in hepatocytes. The binder polypeptide can increase p mRNA by at least twofold, and this increase may persist 3 days, preferably 21 days, after administration (e.g., after a single intraperitoneal dose of 10 mg / kg). The binder polypeptide can decrease serum iron concentration in mice, which may be detectable within 24 hours and preferably persist 3 days, preferably 21 days, after administration. Example experiments and protocols for quantifying serum iron and TSAT to measure hamp mRNA are shown in the examples.

[0195] Binder polypeptide The 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, including conventional IgG antibodies and other binding proteins based on immunoglobulin domains, are known in the art (see Binz, Amstutz, and Pluckthun, Nature Biotechnology 23(10):1257, 2005). Non-immunoglobulin-bound molecules are also known, and binding loops can be created by manipulation within other polypeptide scaffolds such as fibronectin.

[0197] Preferably, the binder polypeptide of the present invention comprises an immunoglobulin domain whose binding site to 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 an immunoglobulin domain, which is either naturally occurring or partially or entirely produced by synthesis. Antibodies may be IgG, IgM, IgA, IgD, or IgE molecules, or antigen-specific antibody fragments thereof (including, but not limited to, Fab, F(ab')2, Fv, disulfide-bonded Fv, scFv, single-domain antibodies, closed-constitution multispecific antibodies, disulfide-bonded scfv, and diabodies), which may be derived from any species that naturally produces antibodies or created 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 includes an antigen-binding site. The antigen-binding site (paratope) is a part of the 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 have residues that directly contribute to the affinity of the interaction. Epitopes can also be three-dimensional, i.e., they can consist of nonlinear amino acids. In certain embodiments, epitopes may contain antigenic determinants, which are chemically active molecular surface groups such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, they may have specific three-dimensional structural features and / or specific charge features.

[0200] The antigen-binding site is a polypeptide or domain that contains one or more CDRs of the antibody and is capable of binding 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 antibody's variable domains.

[0201] The antigen-binding site of an antibody is provided by one or more antibody variable domains. For 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, The binding site includes a VH / VL pair, or two or more such pairs. Therefore, the antigen-binding site of an antibody may include VH and VL.

[0202] An antibody can be an entire immunoglobulin including its 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 region and / or variable region of the intact antibody. Examples of antibody fragments include: (i) Fab fragments, i.e., monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragment, i.e., a divalent fragment containing two Fab fragments linked by disulfide bonds in the hinge region; (iii) Fd fragment consisting of VH and CH1 domains; (iv) Fv fragment consisting of the VL and VH domains of a single arm of the antibody, (v) dAb fragments consisting of VH or VL domains (the whole fragment is incorporated herein by reference, Ward et al. (1989) Nature 341:544~546); and (vi) Isolated complementarity-determining regions (CDRs) that retain specific antigen-binding functionality. These are some examples.

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

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

[0205] In some cases, the binder polypeptide, or its antibody immunoglobulin domain, can be fused or conjugated to a further polypeptide sequence and / or to a label, tag, toxin, or other molecule. 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 may be a multispecific antibody, such as a bispecific antibody, comprising (i) an antigen-binding site of the antibody to MTP-2, and (ii) a further antigen-binding site that recognizes another antigen (in some cases, an antigen-binding site of the antibody described herein).

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

[0207] The antibody may contain the VH domain, including CDR1, CDR2, and CDR3 of VH, as well as the framework. The antibody may also contain, or further, CDR1, CD of VL. The antibody VL domain may include R2 and CDR3, as well as a framework. 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, CDR sets, and HCDR sets and LCDR sets disclosed herein are aspects and embodiments of the present invention. Where described herein, “CDR set” includes CDR1, CDR2, and CDR3. Thus, HCDR set refers to HCDR1, HCDR2, and HCDR3, and LCDR set refers to LCDR1, LCDR2, and LCDR3. Unless otherwise stated, “CDR set” includes HCDR and LCDR.

[0208] As described in more detail in the examples, the inventors have used 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 for specific purposes, referred to as NORI-018, NORI-019, NORI-020, NORI-021, NORI-022, NORI-023, NORI-024, NORI-025, NORI-026, NORI-027, NORI-028, NORI-029, NORI-030, NORI-031, NORI-032, and NORI-033 ("NORI-001 to NORI-033"), were isolated and characterized.

[0209] In various embodiments of the present invention, unless otherwise specified in the context, 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 listings and / or drawings, as well as antibodies having the HCDR and / or LCDR of these antibodies, and optionally having the entire heavy chain amino acid sequence and / or the entire light chain amino acid sequence 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 within the variable domain, or to CDRs or framework regions, refer to the IMGT definition.

[0212] If the VH or VL domain of an antibody contains one or more residues in the framework region that differ from the germline gene segment from which it was obtained by recombination, the non-germline residues may be retained or mutated to different residues, for example, non-germline residues may revert to germline residues. The corresponding germline gene segment is identified as the gene segment to which the sequence of the variable domain is most closely aligned, and the germline gene segments corresponding to the VH and VL domains NORI-001 to NORI-033 are shown herein in Table G.

[0213] An antibody according to the present invention may comprise one or more CDRs described herein, for example, CDR3, and may also comprise CDR1 and CDR2 to form a set of CDRs. A CDR or set of CDRs may be any of the CDRs or set of CDRs from NORI-001 to NORI-033.

[0214] The present invention relates to HCDR1, HCDR2, and / or HCDR3 of any of the antibodies NORI-001 to NORI-033, and / or L of any of these antibodies. We provide an antibody containing, for example, a set of CDRs: CDR1, LCDR2, and / or LCDR3. The antibody may contain a set of VH CDRs from one of these antibodies. In some cases, the antibody may also contain a set of VL CDRs from one of these antibodies, where the VL CDRs are VH It may originate from the same or different antibodies as CDR.

[0215] VH domains containing the disclosed HCDR pair, and / or VL domains containing the disclosed LCDR pair, are also provided by the present invention.

[0216] As will be discussed further below, VH or VL domains alone can be used to bind to an antigen, but typically, a VH domain pairs with a VL domain to create an antigen-binding site on an antibody. The VH domain of NORI-003 can pair with the VL domain of NORI-003, thus forming an antigen-binding site on an antibody that includes both the VH and VL domains of NORI-003. Similar embodiments are provided for other VH and VL domains disclosed herein. In other embodiments, NORI-003 VH pairs with a VL domain other than NORI-003 VL. Indiscriminate pairing of light chains is well known in the art. Here again, similar embodiments are provided by the present invention for other VH and VL domains disclosed herein.

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

[0218] An antibody may contain one or more CDRs, e.g., a set of CDRs, within its antibody framework. The framework region may be a human germline gene segment sequence. Therefore, an antibody may be a human antibody having a VH domain containing a set of HCDRs in a human germline framework. Typically, an antibody may also have a VL domain containing a set of LCDRs in a human germline framework, e.g., a human germline framework. The “gene segment” of an antibody, e.g., the VH gene segment, D gene segment, or JH gene segment, refers to an oligonucleotide having the nucleic acid sequence from which that portion of the antibody originated; for example, the VH gene segment is an oligonucleotide containing a nucleic acid sequence corresponding to a portion of the FR1 to CDR3 of the polypeptide VH domain. Human V, D, and J gene segments can be recombined to produce a VH domain, and human V and J segments can be recombined to produce a VL domain. The D domain or D region refers to the diversity domain or diversity region of the antibody chain. The J domain or J region refers to the linking domain or linking region of the antibody chain. Somatic hypermutations can produce antibody VH or VL domains with framework regions that do not precisely match or align with the corresponding gene segment; however, sequence alignment can be used to identify the nearest gene segment, and consequently, to identify which particular combination of gene segments from which a particular VH or VL domain originates. When aligning an antibody sequence with a gene segment, either 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 directly aligned with the nucleotide sequence of the gene segment.

[0219] The antibody of the present invention may be a human antibody, or a chimeric antibody comprising a human variable region and a non-human (e.g., mouse) constant region. The antibody of the present invention may, for example, have a human variable region and optionally a human constant region.

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

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

[0222] The constant region of the antibody of the present invention may be a non-human constant region. For example, when the antibody is produced in a transgenic animal (examples of which are described elsewhere in this specification), a chimeric antibody containing a human variable region and a non-human (host animal) constant region is produced. Some transgenic animals produce completely human antibodies. Others are engineered to produce antibodies containing a chimeric heavy chain and a completely human light chain. If the antibody contains one or more non-human constant regions, such substitutions may be made to produce antibodies more suitable for administration to humans as a therapeutic composition, as replacing these with human constant regions reduces their immunogenicity.

[0223] Digestion of an antibody with the enzyme papain yields two identical antigen-binding fragments, also known as “Fab” fragments, and an “Fc” fragment that lacks antigen-binding activity but possesses crystallization ability. As used herein, “Fab” refers to an antibody fragment containing one constant domain and one variable domain, each of the heavy and light chains. The term “Fc region” as used herein is used to define the C-terminal region of the immunoglobulin heavy chain, including the native sequence Fc region and variant Fc regions. “Fc fragment” refers to the carboxyl-terminal portions of both H chains held together by a disulfide. The effector function of an antibody is determined by the sequence in the Fc region, which is also recognized by the Fc receptor (FcR) found in certain types of cells. Digestion of an antibody with the enzyme pepsin yields an F(ab')2 fragment, in which the two arms of the antibody molecule remain bound and which contains two antigen-binding sites. The F(ab')2 fragment has the ability to crosslink antigens.

[0224] As used herein, "Fv" refers to the smallest fragment of an antibody that possesses both an antigen recognition site and an antigen binding site. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain, strongly linked by non-covalent or covalent bonds. In this three-dimensional structure, the three CDRs of each variable domain interact to define the antigen binding site on the surface of the VH-VL dimer. Together, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of the Fv containing only three antigen-specific CDRs) has the ability to recognize and bind to an antigen, albeit with lower affinity than the entire binding site.

[0225] The antibodies disclosed herein are modified to increase or decrease their serum half-life. In one embodiment, one or more of the following mutations: T252L, T254S, or T256F are introduced to increase the biological half-life of the antibody. The biological half-life can also be increased by modifying the CH1 domain or CL domain of the heavy chain constant region to have a salvage receptor-binding epitope obtained from the two loops of the CH2 domain of the Fc region of IgG, as described in U.S. Patent No. 5,869,046 and U.S. Patent No. 6,121,022, whose modifications described herein are incorporated herein by reference. 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, resulting in impaired SpA binding in the antibody or fragment compared to the native Fc-hinge domain's SpA binding. Other methods for increasing the serum half-life are known to those skilled in the art. Therefore, 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 yet another embodiment, the antibody or fragment is PAS-modified (i.e., genetically fused to a polypeptide sequence consisting of PAS (XL-Protein GmbH) that forms an uncharged random coil structure with a large hydrodynamic volume). In yet another embodiment, the antibody or fragment is XTEN-modified (registered trademark) / rPEG-modified (i.e., genetically fused to a therapeutic peptide with an inaccurate repeat peptide sequence (Amunix, Versartis)). In yet another embodiment, the antibody or fragment is ELP-modified (i.e., genetically fused to an ELP repeat sequence (PhaseBio)). These various half-life-extending fusions are described in more detail in Strohl, BioDrugs (2015) 29:215-239, and these fusions in Tables 2 and 6, for example, are incorporated herein by reference.

[0226] Antibody constant region As mentioned above, antibodies are offered in various isotypes and with different constant regions. The Fc region of an antibody is recognized by the Fc receptor, and its ability to mediate cellular effector functions, including antibody-dependent cell-mediated cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, and antibody-dependent cell phagocytosis (ADCP) activity, is determined. These cellular effector functions involve recruiting Fc receptor-carrying cells to target cell sites and killing the antibody-bound cells.

[0227] In the context of the present invention, it is desirable to avoid cell effector functions such as ADCC, ADCP, and / or CDC. Therefore, 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 is not mediated by ADCC, ADCP, and / or CDC, or they may lack an Fc region or the entire antibody constant region. The antibody may have a constant region with an effector null.

[0228] Antibodies may have heavy chain constant regions that bind to one or more types of Fc receptors but do not induce cellular effector function, i.e., do not mediate ADCC, CDC, or ADCP activity. Such constant regions may be unable to bind to specific Fc receptors involved in eliciting ADCC, CDC, or ADCP activity.

[0229] Antibodies may have a heavy chain constant region that does not bind to the Fcγ receptor. For example, the constant region may contain an "E" mutation, such as the Leu235Glu mutation (i.e., a mutation in which the wild-type leucine residue is replaced by a glutamate residue), known as IgG4-E. Another optional mutation in the heavy chain constant region is the Ser228Pro mutation ("P" mutation), which increases stability by reducing Fab arm exchange. The heavy chain constant region may be 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 inactivated IgG1.

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

[0231] The antibody constant region is manipulated to extend its in vivo half-life. Examples include the "YTE" mutation and other half-life extension mutations (incorporated herein by reference, Dall'Acqua, Kiener, and Wu, JBC 281(33):23514~23524, 2006, and WO02 / 060919). The triple mutation YTE is a substitution of three amino acids in the CH2 domain of IgG, and these The mutations provide tyrosine at residue 252, threonine at residue 254, and glutamic acid at residue 256, numbered according to the Kabat EU index. As described in the referenced publication, the YTE modification increases the half-life of the antibody compared to the half-life of the corresponding antibody having a human CH2 wild-type domain. To increase the in vivo shelf life, the antibody of the present invention may contain an antibody constant region (e.g., IgG constant region, e.g., IgG CH2 domain) having one or more mutations that increase the half-life of the antibody compared to the corresponding wild-type human constant region (e.g., IgG, e.g., IgG CH2 domain). The half-life can be determined by standard methods such as those described in WO02 / 060919.

[0232] The appropriate antibody constant region can be selected according to the genotype of the patient being treated. For example, as described in US20160319017, a method for increasing erythropoiesis in human patients is: rs855791, rs2543519, rs2235324, and rs1421312 The step may include 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 following; The binder is (a) The heavy chain constant region of human gamma-4, including Leu at position 189 as indicated by SEQ ID NO: 73 of US20160319017, or Arg at position 289 as indicated by SEQ ID NO: 73; and (b) The heavy chain constant region of human gamma-1, including Asp corresponding to position 204 of sequence number 42 in US20160319017, or Leu corresponding to position 206 of sequence number 42 in US20160319017. It includes a steady region selected from the group consisting of; where, (i) The human subject comprises a TMPRSS6 nucleotide sequence containing the selected SNP; and (ii) A human patient has a constant region gene segment that encodes the selected constant region; or a human patient expresses an antibody that contains the selected constant region.

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

[0234] Table S shows further examples of steady-state regions.

[0235] Binder polypeptide generation and modification Methods for identifying and producing antibody-containing binder polypeptides are well known in the art.

[0236] For example, antibodies are produced using experimental animals, such as mice (e.g., Omnirat®), rats (e.g., Omnirat®), camelids, sharks, rabbits, chickens, or other non-human animals, which are immunized with MTP-2 or a fragment thereof (e.g., recombinant MTP-2 ECD) or its encoding nucleic acid, and then optionally humanized to produce human or humanized antibodies by humanizing the constant and / or variable regions. In one example, as will be apparent to those skilled in the art, display techniques such as yeast, phage, or ribosome displays are used. For example, standard affinity maturation using display techniques is performed on transgenic animals, phage displays, etc. This is carried out in a further step after isolating antibodies derived from an or other library. A representative example of a suitable technique is described in the method presented in paragraphs

[0309] to

[0346] , for example, of US20120093818 (Amgen, Inc.), which is incorporated herein by reference in its entirety.

[0237] There are many reasons why it may be desirable to create binder variants, including optimizing polypeptide sequences for large-scale production, facilitating purification, enhancing stability, or improving suitability for encapsulation in desired pharmaceutical formulations. For example, protein engineering work is performed on one or more target residues of an antibody sequence, such as substituting one amino acid with another (with the exception of Cys and Met, but in some cases generating variants containing all naturally occurring amino acids at this position) 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 be undesirable because doing so could cause problems in production, for example, by forming new intramolecular or intermolecular cysteine-cysteine ​​bonds. Once a primary candidate has been selected and optimized for production and clinical development, it is generally desirable to alter its antigen-binding properties as little as possible, or at least retain the affinity and titer of the parent molecule. However, variants can also be generated to modulate important antibody characteristics such as affinity, cross-reactivity, or neutralizing titer.

[0238] An antibody may contain one or more H and / or L CDRs in any of the disclosed antibody sets, each containing one or more amino acid mutations in the disclosed set of H and / or L CDRs. The mutations may be amino acid substitutions, deletions, or insertions. For example, there may be one or more amino acid substitutions in the disclosed set of H and / or L CDRs. For example, there may be up to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 mutations, e.g., substitutions, in a set of H and / or L CDRs. For example, HCDR3 may contain up to 6, 5, 4, 3, or 2 mutations, e.g., substitutions, and / or LCDR3 may contain up to 6, 5, 4, 3, or 2 mutations, e.g., substitutions. The antibody may comprise a set of HCDRs, LCDRs, or a set of six (H and L)CDRs as shown for any NORI antibody herein, or a set of CDRs comprising one or two conservative substitutions.

[0239] One or more amino acid mutations may be introduced into the framework region of the antibody VH or VL domain disclosed herein. For example, one or more residues different from the corresponding human germline segment sequence are reintroduced into the germline. The 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 antibodies may include 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 listed in the attached sequence listing, and / or 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 percentage of identity between two amino acid sequences include, for example, BLAST, FASTA, or the Smith-Waterman algorithm using, for example, default parameters. A particular variant may include one or more amino acid sequence changes (addition, deletion, substitution, and / or insertion of amino acid residues).

[0241] The changes affect one or more framework areas and / or one or more of them. The above modifications are performed on CDRs. Mutations are sometimes provided by CDR mutagenesis. The modifications usually do not result in loss of function; therefore, antibodies containing such modified amino acid sequences may retain their ability to bind to MTP-2. Such antibodies may retain the same quantitative binding ability as unmodified antibodies, for example, when measured in the assays described herein. Antibodies containing such modified amino acid sequences may have improved ability to bind to and / or inhibit MTP-2.

[0242] Modifications may include replacing one or more amino acid residues with naturally absent or non-standard amino acids, modifying one or more amino acid residues to naturally absent or non-standard forms, or inserting one or more naturally absent or non-standard amino acids into the sequence. Examples of the number and location of modifications in the sequence of the present invention are described elsewhere in the invention. Naturally occurring amino acids include 20 “standard” L-amino acids, identified by standard single-letter notation as G, A, V, L, I, M, P, F, W, S, T, N, Q, Y, C, K, R, H, D, E. Non-standard amino acids include any other residues that may be incorporated into the polypeptide backbone or result from modifications of existing amino acid residues. Non-standard amino acids may or may not be naturally occurring.

[0243] As used herein, the term “mutant” 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 nevertheless retains one or more specific functions or biological activities of the parent molecule. Amino acid substitutions include modifications in which an amino acid is replaced with a different naturally occurring amino acid residue. Such substitutions are classified as “conservative,” in which case the amino acid residue contained in the polypeptide is replaced with another naturally occurring amino acid having similar characteristics in terms of polarity, side-chain functionality, or size. Such conservative substitutions are well known in the art. Substitutions encompassed by the present invention may also be “non-conservative,” in which an amino acid residue contained in a peptide is replaced with an amino acid having different properties, for example, a naturally occurring amino acid from a different group (e.g., a charged or hydrophobic amino acid replaced with alanine), or a naturally occurring amino acid is replaced with a novel amino acid. In some embodiments, the amino acid substitutions are conservative. Furthermore, when used in relation to polynucleotides or polypeptides, the polynucleotides or polypeptides included within the term variant refer to polynucleotides or polypeptides that may have different primary, secondary, or tertiary structures compared to a reference polynucleotide or polypeptide (e.g., compared to a wild-type polynucleotide or polypeptide).

[0244] In some embodiments, “synthetic mutants,” “recombinant mutants,” or “chemically modified” polynucleotide or polypeptide mutants isolated or generated using methods known in the art may be used. “Modified mutants” may include conserved or non-conserved amino acid changes, as described below. Polynucleotide changes can result in substitution, addition, deletion, fusion, and truncation of amino acids in the polypeptide encoded by the reference sequence. Some embodiments use insertion mutants, deletion mutants, or substitution mutants by amino acid substitution, including insertions and substitutions of amino acids and other molecules not typically occurring in the underlying peptide sequence of the mutant, such as, but not limited to, insertions of ornithine not typically occurring in human proteins. The term “conservative substitution,” when referring to polypeptides, means a change in the amino acid composition of a polypeptide that does not substantially alter the polypeptide’s activity. For example, a conservative substitution refers to substituting an amino acid residue for a different amino acid residue having similar chemical properties (e.g., acidic, basic, positively or negatively charged, polar or nonpolar, etc.). Conservative amino acid substitutions include replacing leucine with isoleucine or valine, replacing aspartic acid with glutamic acid, or replacing threonine with serine. Conservative substitutions provide functionally similar amino acids. The table is 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), which is incorporated by reference in its entirety). In some embodiments, individual substitutions, deletions or additions that change, add or delete a single amino acid or a small percentage of amino acids can also be considered "conservative substitutions" if the change does not reduce the activity of the peptide. Insertions or deletions are typically in the range of about 1 to 5 amino acids. The choice of conservative amino acids is based on the position of the amino acid being substituted in the peptide, for example, whether the amino acid is on the outside of the peptide and exposed to the solvent or on the inside and not exposed to the solvent.

[0245] Based on the position of the existing amino acid, including exposure to the solvent (i.e., whether the amino acid is exposed to the solvent or present on the outer surface of the peptide or polypeptide compared to an amino acid that is localized internally and not exposed to the solvent), an amino acid that substitutes for the existing amino acid can be selected. The choice of such conservative amino acid substitutions can be found, for example, in Dordo et al., J. It is well known in the art as disclosed in MoI Biol, 1999, 217, pp. 721-739, and Taylor et al., J. Theor. Biol. 119(1986); pp. 205-218, and S. French and B. Robson, J. Mol. Evol. 19(1983)171. Thus, conservative amino acid substitutions suitable for the outer amino acids of a protein or peptide (i.e., the amino acids exposed to the solvent) can be selected, for example, but not limited to, the following substitutions are used: substitution of Y by F, substitution of T by S or K, substitution of P by A, substitution of E by D or Q, substitution of N by D or G, substitution of R by K, substitution of G by N or A, substitution of T by S or K, substitution of D by N or E, substitution of I by L or V, substitution of F by Y, substitution of S by T or A, substitution of R by K, substitution of G by N or A, substitution of K by R, substitution of A by S, K or P.

[0246] In alternative embodiments, conservative amino acid substitutions can also be selected that are suitable for the inner amino acids of a protein or peptide, for example, conservative substitutions can be used that are appropriate for amino acids inside a protein or peptide (i.e., amino acids not exposed to the solvent), for example, but not limited to, the following conservative substitutions can be used: Y is substituted by F, T is substituted by A or S, I is substituted by L or V, W is substituted by Y, M is substituted by L, N is substituted by D, G is substituted by A, T is substituted by A or S, D is substituted by N, I is substituted by L or V, F is substituted by Y or L, S is substituted by A or T, and A is substituted by S, G, T or V. In some embodiments, non-conservative amino acid substitutions are also included within the term variant.

[0247] The present invention includes a method of making an antibody containing VH and / or VL domain variants of the antibody VH and / or VL domains shown in Table S. Such antibodies are (i) The parent antibody VH domain is a VH domain containing the VH domain of any of NORI-001 to NORI-033, or the heavy chain complementarity determining region of any of those antibodies. A step of providing an antibody VH domain, which is an amino acid sequence variant of the 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) A step of providing a VH / VL combination by combining the VH domains provided in this manner with VL domains as appropriate, (iii) A step of testing the VH domain or VH / VL domain combination thus provided to identify an antibody having one or more desired features. It can be produced by a method that includes [a specific method].

[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] Desired features include binding to human and / or non-human MTP-2. Antibodies with equivalent or higher affinity to human and / or mouse MTP-2 compared to the parent antibody are identified. Other desired features include inhibition in the enzyme assays described herein, as well as in vivo reduction of serum iron concentration and / or TSAT, and increase of hamp mRNA. Identification of antibodies with desired features may include identification of antibodies with functional attributes described herein, such as affinity, cross-reactivity, specificity, or neutralizing titer, all of which can be determined by the assays described herein.

[0250] If a VL domain is included in the method, the VL domain may be any VL domain from NORI-001 to NORI-033, or a variant provided by the addition, deletion, substitution, or insertion of one or more amino acids into the amino acid sequence of the parent VL domain, the parent VL domain being any VL domain from NORI-001 to NORI-033, or a VL domain containing the light chain complementarity determining region of any of those 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] A method for generating a mutant antibody may optionally include a step of preparing a copy of the antibody or VH / VL domain combination. The method may further include a step of expressing the resulting antibody. The nucleotide sequences corresponding to the desired antibody VH and / or VL domains can optionally be prepared using one or more expression vectors. Appropriate methods of expression, including recombinant expression in host cells, are described in detail herein.

[0252] Encoding nucleic acids and methods for producing them 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 of synthetic origin, or any combination thereof, can encode an 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. References to nucleotide sequences described herein encompass DNA molecules having the given sequence and RNA molecules having the given sequence with U substituted in place of T, unless the context specifically requires otherwise.

[0254] The present invention also provides recombinant host cells containing one or more nucleic acids encoding the antibody. Methods for producing the encoded antibody may include, for example, expression from nucleic acids by culturing recombinant host cells containing nucleic acids. The antibody is therefore obtained using any suitable method, isolated and / or purified, and then used as needed. The production method involves a composition comprising at least one additional component, such as a pharmaceutically acceptable excipient. The process may include the step of formulating the substance into a physical product.

[0255] Systems for the cloning and expression of polypeptides in various different host cells are well known. Suitable host cells include bacteria, mammalian cells, plant cells, filamentous fungi, yeast and baculovirus systems, as well as transgenic plants and animals.

[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 under culture is also available to those skilled in the art as an option for production. Mammalian cell lines available in the art for the 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 retinal cells, and many other cells.

[0257] The vector may contain appropriate regulatory sequences, including, as necessary, promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes, and other sequences. A nucleic acid encoding an antibody is introduced into a host cell. The nucleic acid of the present invention can be incorporated into the genome (e.g., chromosomes) of the host cell. Integration can be facilitated by including sequences that promote recombination with the genome, according to standard techniques. The nucleic acid can be introduced into eukaryotic cells by various methods, including calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses, e.g., vaccinia or, in insect cells, baculoviruses. The introduction of the nucleic acid into host cells, particularly eukaryotic cells, may be done using virus or plasmid-based systems. Plasmid systems are maintained as episomes or incorporated into host cells or artificial chromosomes. Integration may be by either random or targeted integration of one or more copies at a single or multiple loci. With regard to bacterial cells, appropriate techniques include calcium chloride transformation, electroporation, and transfection using bacteriophages. Following transfection, host cells may be cultured under conditions for gene expression to express nucleic acids, and then, optionally, binder polypeptides, such as antibodies, may be isolated or purified.

[0258] Formulation and administration The binder polypeptides and their coding nucleic acid molecules according to the present invention will typically be provided in isolated form. The VH and / or VL domains, as well as the nucleic acids, will be provided purified from their natural environment or the environment in which they are produced. The isolated binder polypeptides and isolated nucleic acids will not contain, or substantially contain, the substances to which they naturally associate, e.g., other polypeptides or nucleic acids found together with them in vivo, or the environment in which they are produced (if such production is by recombinant DNA technology in vitro) (e.g., cell cultures). In some cases, the isolated binder polypeptide or nucleic acid will (1) not contain at least some other proteins that are typically found together with it, (2) not contain essentially other proteins from the same source, e.g., from the same species, (3) not be expressed by cells of a different species, (4) be isolated from at least about 50 percent of the polynucleotides, lipids, carbohydrates, or other substances to which it naturally associates, (5) operably associate with polypeptides that do not naturally associate (by covalent or non-covalent interactions), or (6) not occur naturally.

[0259] Binder polypeptides or their coding nucleic acids are formulated with diluents or adjuvants and further isolated for practical purposes. For example, if they are used to coat microtiter plates for use in immunoassays. The binder polypeptide is mixed with a carrier, and when used therapeutically, it is mixed with a pharmaceutically acceptable carrier or diluent. Other active ingredients may also be included in the therapeutic product, as described elsewhere in the present invention. The binder polypeptide is glycosylated naturally in vivo or by a system of heterologous eukaryotic cells such as CHO cells, or (for example, produced by expression in prokaryotic cells) is deglycosylated. The present invention encompasses antibodies having modified glycosylation patterns.

[0260] Typically, the isolated product constitutes at least about 5%, at least about 10%, at least about 25%, or at least about 50% of a given sample. The binder polypeptide shall not substantially contain any proteins or polypeptides or other contaminants found in its natural or production environment that would interfere with its therapeutic, diagnostic, prophylactic, research, or other use.

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

[0262] The composition may contain suitable carriers, excipients, and other agents incorporated into the formulation to effect improvements in movement, delivery, tolerability, etc. Numerous suitable formulations can be found in formularies known to all pharmacists: Remington's Pharmaceutical 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 absorption pastes, oil - in - water and water - in - oil emulsions, emulsion carbowaxes (polyethylene glycols of various molecular weights), semi - solid gels, and semi - solid mixtures containing carbowaxes. 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 an adjuvant.

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

[0264] A variety of delivery systems are known and can be used to administer the pharmaceutical composition of the present invention. The 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 the epithelial or mucosal - cutaneous layer (such as 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. The administration can also be self - administration by the patient, for example, self - injection.

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

[0266] In certain circumstances, pharmaceutical compositions can be delivered via a sustained-release system. In one embodiment, a pump is used (see Langer, above; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14: p. 201). In another embodiment, a polymer material is used; Medical Applications of Controlled Release, edited by Langer and Wise, CRC See Press., Boca Raton, Fla. (1974). In yet another embodiment, the sustained-release system is placed near the target of the composition and therefore requires only a portion of the systemic dose (see, for example, Medical Applications of Controlled Release, Goodson, Vol. 2, pp. 115-138, 1984).

[0267] The injectable formulations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injection, intravenous infusion, etc. These injectable formulations can be manufactured by known methods. For example, an injectable formulation can be manufactured by dissolving, suspending, or emulsifying the antibody or a salt thereof described above in a sterile aqueous or oily medium conventionally used for injection. Examples of aqueous media for injection include physiological saline, glucose-containing isotonic solutions, and other adjuvants, used in combination with suitable solubilizers such as alcohol (e.g., ethanol), polyhydric alcohol (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Examples of oily media include sesame oil and soybean oil, used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injection thus manufactured can be filled into a suitable ampoule. The pharmaceutical compositions of the present invention can be delivered subcutaneously or intravenously using standard needles and syringes. The treatment is not expected to be limited to use in a clinic. Therefore, subcutaneous injection using needleless devices is also advantageous. With regard to subcutaneous delivery, pen-type delivery devices are readily applicable to the delivery of the pharmaceutical composition of the present invention. Such pen-type delivery devices may be reusable or disposable. Reusable pen-type delivery devices generally utilize replaceable cartridges containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, 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. Disposable pen-type delivery devices do not have replaceable cartridges. Conversely, disposable pen-type delivery devices are designed to be pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is empty of the pharmaceutical composition, the entire device is discarded. Numerous reusable pen-type and auto-injector-type delivery devices are applicable to the subcutaneous delivery of the pharmaceutical composition of the present invention. Examples include: To name just a few examples, there are AUTOPEN (trademark) (Owen Mumford, Inc., Woodstock, UK), DISETRONIC (trademark) pens (Disetronic Medical Systems, Burgdorf, Switzerland), HUMALOG MIX 75 / 25 (trademark) pens, HUMALOG (trademark) pens, HUMALIN 70 / 30 (trademark) pens (Eli Lilly and Co., Indianapolis, Ind.), NOVOPEN (trademark) I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR (trademark) (Novo Nordisk, Copenhagen, Denmark), BD (trademark) pens (Becton Dickinson, Franklin Lakes, NJ), OPTIPENT (trademark), OPTIPEN PRO (trademark), OPTIPEN STARLET (trademark), and OPTICLIKT (trademark) (Sanofi-Aventis, Frankfurt, Germany), but of course, these are not the only examples. Examples of disposable pen-type delivery devices applicable to the 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 manufactured into dosage forms in unit doses suitable for the dosage of the active ingredient. Such dosage forms in unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the aforementioned antibody contained is generally about 5 to about 500 mg per unit dose of dosage form; in particular, in the form of injection, the aforementioned antibody is contained in about 5 to about 100 mg, and in other dosage forms, in about 10 to about 250 mg.

[0269] Binder polypeptides, nucleic acids, or compositions containing the same are contained in medical containers such as vials, syringes, IV containers, or injection devices. In one example, the binder polypeptide, nucleic acid, or composition may be in vitro and contained in a sterile container. In one example, a kit is provided comprising a binder polypeptide, packaging, and instructions for use in the therapeutic method described herein.

[0270] One aspect of the present invention is a composition comprising the binder polypeptide or nucleic acid of the present invention and one or more pharmaceutically acceptable excipients, as examples are listed above. "Pharmaceutically acceptable" means that it is approved or expected to be approved by a U.S. federal or state regulatory authority for use in animals, including humans, or that it is listed in the United States Pharmacopeia or any other generally accepted pharmacopoeia. A pharmaceutically acceptable carrier, excipient, or adjuvant can be administered to a patient together with the binder polypeptide, for example, any antibody or polypeptide molecule described herein, without impairing its pharmacological activity and is non-toxic when administered in a dose sufficient to deliver a therapeutic dose of the drug.

[0271] In some embodiments, the binder polypeptide will be the sole active ingredient in the composition according to the present invention. Therefore, the composition may consist of an antibody, or it may consist of a binder polypeptide together with one or more pharmaceutically acceptable excipients. However, the composition according to the present invention may optionally contain one or more additional active ingredients. Other therapeutic agents that may be desirable to administer together with the binder polypeptide or nucleic acid according to the present 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 the binder polypeptide or nucleic acid according to the present invention, whether as a combination formulation or as separate formulations, or may be provided in a composition together with the binder polypeptide or nucleic acid according to the present invention. The binder polypeptide or nucleic acid according to the present invention may be administered separately and sequentially, or simultaneously and optionally as a combination formulation, with other therapeutic agents such as those mentioned. It is possible.

[0272] Multiple compositions may 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 at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12 hours apart. Compositions may also be administered at intervals of 24 hours or longer. Alternatively, two or more compositions may be administered simultaneously, for example, at intervals of less than 10 minutes or less than 5 minutes. In some embodiments, simultaneously administered compositions may be administered as a mixture by similar or different time-release mechanisms for each component, or without such mechanisms.

[0273] Binder polypeptides and their coding nucleic acids can be used as therapeutic agents. Patients as used herein are generally mammals, typically humans. Binder polypeptides or nucleic acids can be administered to mammals, for example, by any of the administration routes described herein. In preferred embodiments, binder polypeptides are administered by subcutaneous injection.

[0274] The dose is typically the "therapeutic effective dose," which is the amount administered to produce the desired effect, sufficient to demonstrate a benefit to the patient. The exact amount depends on the purpose of the treatment and can be determined by those skilled in the art using known methods (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding). The determination of the prescription of a treatment, such as the dosage, is the responsibility of the general practitioner and other physicians and may depend on the severity of the symptoms and / or progression of the disease being treated. The therapeutic effective dose 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 doses to humans from mice and other test animals are known.

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

[0276] As used herein, the terms “treat,” “treating,” or “amelioration” refer to therapeutic interventions aimed at reversing, reducing, improving, inhibiting, slowing, or stopping the progression or severity of a condition associated with a disease or disorder. The term “treating” includes reducing or mitigating at least one adverse effect or symptom of a condition, disease, or disorder. A treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Or, a treatment is “effective” if the progression of the disease is reduced or stopped. That is, “treatment” includes not only improvement of symptoms or markers but also cessation, or at least slowing, of the progression or worsening of symptoms compared to what would be expected in the absence of the treatment. Beneficial or desired clinical outcomes include, but are not limited to, the reduction of one or more symptoms, whether detectable or undetectable; a reduction in the severity of the disease; stabilization of the disease state (i.e., no worsening); delay or slowing of disease progression; improvement or mitigation of the disease state; remission (whether partial or complete remission); and / or a reduction in mortality. The term “treatment” of the disease also includes providing relief from the symptoms or side effects of the disease (including palliative treatment). With respect to effective treatment, complete cure is not intended. The method is, in some cases, particularly In certain embodiments, this may include healing. In the context of the present invention, the treatment may be a preventive treatment.

[0277] A long half-life is a desirable feature of the binder polypeptide of the present invention. An extended half-life leads to a reduction in administration frequency, resulting in fewer injections required to maintain therapeutically effective concentrations of the molecule in the bloodstream. The in vivo half-life of the antigen-binding molecule 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 molecule 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] A binder polypeptide is provided to be administered at regular intervals of one week, two weeks, three weeks, four weeks, or one month.

[0279] therapeutic use Therapeutic applications addressed by this invention include the treatment of patients for whom inhibiting MTP-2 activity and / or reducing iron uptake would be beneficial. A specific therapeutic area is anemia characterized by high iron load and deposition as well as insufficient erythropoiesis. As previously discussed, several forms of anemia are characterized by primary iron overload (caused by inappropriately low hepcidin levels due to high erythropoietic activity) and ineffective erythropoiesis, which often occurs in association with secondary iron overload, mainly due to repeated red blood cell transfusions. Iron overload contributes to anemia by negatively impacting organ function, negatively affecting erythropoiesis, increasing hemiclomes and ROS in erythroid progenitor cells, driving apoptosis, and resulting in a near-total loss of functional red blood cells. Reduced iron reduces apoptosis, improves alpha / betaglobin imbalance, and allows for the production of more mature red blood cells. Reduced iron utilization by erythroid cells leads to decreased heme production, increased maturation of erythroid precursors, and increased hemoglobin levels. Therefore, in some conditions, the therapeutic mechanism of action of binder polypeptides may be the inhibition of the enzymatic activity of MTP-2, which increases hepcidin levels, leading to iron restriction that normalizes erythrocyte production and improves hemoglobin and erythrocyte quality.

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

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

[0282] The treatment may be beneficial in many conditions, including those discussed herein: Myelodysplastic syndromes (MDS) with a lower risk of ring sideroblasts (RARS) requiring blood transfusions 5q-MDS; Transfusion-dependent beta-thalassemia or severe beta-thalassemia; Transfusion-independent beta-thalassemia or intermediate-type beta-thalassemia; For example, hemochromatosis in patients without HJV or hepcidin mutations, or hemochromatosis type 1 or 3; Cirrhosis of the liver; fatty liver; For example, hepatic fibrosis in patients with NASH (non-alcoholic steatohepatitis) or ASH; Blackfan Diamond Anemia; Pulmonary arterial hypertension; Anemia in sickle cell disease ("sickle cell anemia"); polycythemia vera; Chronic kidney disease-associated anemia (CKD).

[0283] Therefore, patients treated by the present invention may have one of the above conditions. Treatment methods may include administering a binder polypeptide, nucleic acid, or composition described herein to the patient. Examples of formulations and methods of administration are described elsewhere in the present invention. Treatment, depending on the disease condition, may begin after diagnosis, shortly after birth, or with the onset of transfusion dependence or an increase in serum ferritin, or otherwise when iron chelation is initiated.

[0284] Treatment with anti-MTP-2 binder polypeptides is thought to increase hepcidin levels, leading to a decrease in TSAT levels accompanied by reduced heme and hemimicrome 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. While single-cell hemoglobin content would decrease, the increased red blood cell count would result in higher total hemoglobin levels. This, in turn, would 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., extend the survival of treated patients. Avoiding or reducing the burden of blood transfusions, and / or avoiding or reducing the need for iron chelation and / or venotomy, should also improve the patient's quality of life.

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

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

[0288] Inhibition of MTP-2 may also be useful in treating 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):1350p). 2018).

[0289] Ideally, effective treatment for the patient should be achieved without serious adverse effects. Side effects should be absent or only mild.

[0290] Treatment using the binder polypeptide according to the present invention can be combined with one or more additional treatments, for example, further therapeutic agents for treating iron overload. The inhibitor polypeptide can be combined with activin type II receptor agonist fusion proteins, such as ruspatercept. MTP-2 inhibitors may offer synergistic effects on erythropoiesis because they represent an alternative mechanism of action to other planned and existing therapies. Combinations of (i) MTP-2 inhibitors and (ii) TGFβ superfamily ligand antagonists, such as TGFβ superfamily receptor-based ligand scavenger / traps, may therefore yield favorable 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 protein (BMP). Receptor-ligand trap molecules are constructed by providing the extracellular domain of the receptor in a soluble form, where the receptor retains its ability to bind to its ligand but does not induce downstream signaling (otherwise resulting from normal receptor-ligand interactions). The receptor extracellular domain can be ligated to the Fc region to form fusion proteins.

[0292] Preferably, the receptor is an activin II receptor, e.g., ActIIRA or ActIIRB. The antagonist may be a polypeptide containing the soluble extracellular domain of an activin II receptor fused to the Fc region, e.g., activin receptor IIB (ActRIIB). Ruspatercept is one such molecule and is currently used to treat anemia in beta-thalassemia and myelodysplastic syndromes. Examples of ActRIIA and ActRIIB ligand traps can also be found in US7988973 of Acceleron Pharma, incorporated herein by reference. Suragani RN et al. previously described a modified human ActRIIB extracellular domain (residues 24-131 of native progenitor cells with the L79D substitution) "RAP-536" linked to the mouse IgG2a Fc domain, which was reported to reduce ineffective erythropoiesis and disease complications in mouse β-thalassemia (Blood 123(25):3864-3872 2014). Sotatercept is an example of an activin type II A receptor IgG-Fc fusion protein. Antagonists of other TGFβ family ligands, such as BMPR-Fc fusion proteins, can be similarly generated.

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

[0294] Antagonists of TGFβ family ligands, such as ActRIIB-Fc, ActIIRIIA-Fc, or BMPR-Fc, promote the maturation of erythrocyte progenitor cells during erythropoiesis, while MTP-2 inhibitors lead to iron reduction (helping to normalize pathologically high iron levels) and thus delay erythropoiesis. From the studies reported herein (see Example 22), we believe that a combination of these two actions results in more efficient production of more mature erythrocytes. We demonstrate, for example, that a combination of an MTP-2 inhibitor (represented by NORI-11-M) and an activin receptor II ligand trap (represented by ActRIIB-Fc) is more effective than the ligand trap alone in correcting both iron overload and associated anemia. Combinations with MTP-2 inhibitors may expand the therapeutic potential of TGFβ superfamily ligand traps by providing greater therapeutic efficacy in patients for whom TGFβ superfamily ligand traps are already indicated (e.g., non-transfusion-dependent β-thalassemia patients). Combinations with MTP-2 inhibitors may also expand the therapeutic potential of ligand traps to treat additional patient groups, such as patients with severe β-thalassemia, for whom treatments such as ruspatercept currently offer limited benefits.

[0295] Ruspatercept was recently approved by the FDA for myelodysplastic syndrome. Iron overload begins before MDS patients become transfusion-dependent because ineffective erythropoiesis suppresses hepcidin production in the liver, and therefore leads to unrestricted intestinal iron uptake. Transfusions then exacerbate the iron overload. The same situation occurs in transfusion-dependent β-thalassemia patients. In such patients treated with ruspatercept (or other TGFβ superfamily ligand traps), the therapeutic benefit will be improved by including an MTP-2 inhibitor in the treatment regimen. Combination therapy can be used for any therapeutic indication or condition described herein, e.g., Blackfan-Diamond anemia.

[0296] The MTP-2 inhibitors used in combination therapy may be the binder polypeptides described herein, or other types of molecules such as nucleic acid inhibitors of TMPRSS6 expression (e.g., antisense or siRNA molecules targeting TMPRSS6) or small molecule inhibitors (e.g., 3-amidinophenylalanine-derived matryptase-1 and -2 protease inhibitors and derivatives). Examples of such inhibitors are 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). Therefore, patients treated by the present invention may also be patients who receive treatment with further therapeutic agents to reduce iron excess. The method may include co-administering the binder polypeptide and the further therapeutic agent to the patient, possibly in separate formulations. The compositions are administered sequentially or concurrently. The same applies when small molecules or nucleic acid MTP-2 inhibitors are used instead of the binder polypeptide. That is, the patient is treated either concurrently or sequentially with a combination of the MTP-2 inhibitor and the further therapeutic agent. The MTP-2 inhibitor and the further therapeutic agent are preferably provided in separate formulations and administered separately. Generally, sequential administration is performed on the same day (sometimes separated by a period of several minutes or hours) or on different days.

[0297] Treatment with MTP-2 inhibitors (e.g., binder polypeptides 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 to standard epo alfa and epo beta proteins. Darbepoetin alfa is used to stimulate red blood cell production in anemic patients to increase hemoglobin levels and reduce the need for blood transfusions. The drug is administered in different doses depending on the severity of anemia, but in patients with chronic kidney disease-associated anemia (CKD), the recommended starting dose is 0.45 mcg / kg iv / sc every four weeks until hemoglobin levels reach over 10 g / dL, at which point the dose is subsequently reduced. ARANESP has been shown to improve heglobin levels in a small-scale study in patients with intermediate-stage beta-thalassemia (Singer et al. 2011), but generally, ARANESP is not a treatment option for patients with beta-thalassemia due to the unacceptable increase in RBC apoptosis and associated splenomegaly. As the results presented herein indicate, therapeutic benefits can be obtained by treating patients with the binder polypeptide and epo according to the present invention. For example, it should be possible to co-administer ARANESP and anti-MTP-2 mAb treatment in humans by two separate sc injections administered simultaneously every 2-4 weeks, based on results obtained in a beta-thalassemia mouse model (see Examples 20 and 21). Such a concurrent treatment strategy should result in a therapeutic improvement in anemia from ARANESP treatment while maintaining spleen size and reducing concurrent toxic iron excess resulting from anti-MTP2 treatment. While the exact combination dosing regimen will be investigated and optimized for humans, it will generally involve the administration of the binder polypeptide according to the present invention to the patient, and similarly, the administration of erythropoietin (preferably recombinant erythropoietin or a medically approved variant thereof, e.g., darbepoetin alfa). In some cases, the binder polypeptide and epo are administered concurrently; or they are administered sequentially (on the same day or on different days). In some cases, the binder polypeptide and epo are administered subcutaneously by separate injections or mixed injections. In some embodiments, the present invention provides treatment of a patient with epo and binder polypeptide, for example, in a patient having a condition described herein such as beta-thalassemia, the administration of the binder polypeptide reduces erythrocyte apoptosis and the accompanying splenomegaly associated with the administration of epo. Binder polypeptides can therefore be used to normalize erythrocyte production in patients undergoing EPO treatment.

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

[0299] [Figure 1]This diagram illustrates the pathways that control the supply of iron for erythrocyte production and other cellular functions. The absorption of dietary iron and the release of iron recovered from aging red blood cells ("aged RBCs") are regulated by hepcidin, which downregulates ferroportin (FPN), an iron transporter protein found in the membranes of intestinal epithelial cells (which take up dietary iron) and macrophages (which take up aging red blood cells). Inhibition of ferroportin reduces iron transport from these cells, thereby limiting the supply of new and recycled iron available to the body. Hepcidin expression from the hamp gene in hepatocytes is regulated by the BMP / SMAD pathway, which is initiated by the binding of its ligand, BMP6, to receptors on the hepatocyte surface (e.g., HJV and BMPR I-II). Negative feedback from elevated serum iron concentrations can upregulate the BMP / SMAD pathway, increasing hepcidin expression and thereby suppressing iron release via ferroportin. Matriptase-2 (MTP-2) is also present on the surface of hepatocytes and likely downregulates the BMP / SMAD pathway by cleaving coreceptors such as HJV, thereby reducing hepcidin expression and allowing for more iron release via ferroportin. [Figure 2] This is a diagram showing the domain structure of MTP-2. [Figure 3] This graph shows the complete dose-response curves of selected anti-MTP-2 antibodies and negative control antibodies in protein-based enzyme assays using (a) human and (b) mouse MTP-2. All antibodies are of the human IgG4PE isotype. [Figure 4] This graph shows the percentage inhibitory effect of antibodies in an enzyme assay using human MTP-2 ECD, plotted against the percentage inhibitory effect of the first and second antibody groups obtained from transgenic mouse immunization in an enzyme assay using mouse MTP-2 ECD. [Figure 5] This graph shows the complete dose-response curves for selected anti-MTP-2 antibodies and negative control antibodies in a cell-based enzyme assay using human MTP-2 ECD. All antibodies are isotype human IgG4PE. [Figure 6] This graph shows the results of an HTRF assay of antibodies that bind 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] This graph shows the results of HTRF assays of antibodies binding to wild-type (a) human, (b) mouse, and (c) cynomolgus monkey MTP-2 ECD. IC = isotype control. All antibodies are human IgG4PE. [Figure 8] This graph shows the results of HTRF competitive assays of selected anti-MTP-2 antibodies or unlabeled aprotinin competing for binding to (a) human and (b) mouse MTP-2 ECD. IC = isotype control against labeled aprotinin-647. All antibodies are human IgG4PE. [Figure 9] This graph shows (a) hamp mRNA levels and (b) serum iron levels from wild-type mice treated with anti-MTP-2 antibody or a control. [Figure 10] This graph shows (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] This graph shows (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] This graph shows (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]This graph shows the time-course evaluation results for (a) HAMP, (b) serum iron, and (c) PK after single IP injections of NORI-008 at 10, 3, and 1 mg / kg into normal mice. [Figure 14] This graph shows the results of the 1-week evaluation of IgG / kappa anti-MTP2 antibody (10 mg / kg IP dose) regarding its effect on reducing serum iron and transferrin saturation. [Figure 15] This graph shows the results of an evaluation of the ability of three anti-MTP-2 antibodies, composed of fully human IgG4 and mouse IgG1, to lower serum iron levels after subcutaneous injection. [Figure 16] This graph shows (a) serum iron concentration and (b) antibody concentration 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] This graph shows the results of the first in vivo rat study in which NORI-008 and NORI-010 were administered as intravenous therapy (IP) at a dose of 10 mg / kg to a group of three wild-type Wistar rats. [Figure 18-1] This graph shows the results of a second in vivo rat study in which NORI-008, NORI-011, NORI-003, and NORI-006 were administered as SQ doses of 10 mg / kg to groups of five wild-type Han Wistar rats. [Figure 18-2] Continuation of Figure 18-1. [Figure 19] This graph shows the results of a two-week study in a thalassemia intermedia Hbbth3 / + mouse model using NORI-010. a) Hepcidin mRNA from liver samples measured by qPCR, b) Serum iron levels [μg / dL] measured by a chromogenic assay, and c) Calculated transferrin saturation [%]. [Figure 20-1]This graph shows the results of an 8-week study of Thalassemia intermedia Hbbth3 / + mouse models using NORI-011-M, in and out of the presence of erythropoietin. a) Hepcidin mRNA from liver samples measured by qPCR, b) Red blood cell count, c) Iron tissue content in liver per gram of wet tissue weight, d) Hemoglobin level, e) Hematocrit, f) Mean corpuscular volume (MCV), g) Red blood cell distribution width (RDW), h) Mean corpuscular hemoglobin (MCH), i) Number of erythroid cells at developmental stages I-V in the spleen, determined by flow cytometry, j) Number of erythroid cells at developmental stages I-V in the bone marrow, determined by flow cytometry, and k) Spleen index. [Figure 20-2] Continuation of Figure 20-1. [Figure 21-1] This graph shows the results of an 8-week study of a thalassemia intermedia Hbbth3 / + mouse model using NORI-011-M, with or without concurrent treatment with ActRIIB-Fc. NORI-011-M was administered once a week at 10 mg / kg, and ActRIIB-Fc was administered twice a week at 10 mg / kg. All samples for readout were obtained at the end of the 8-week study. a) Hepcidin mRNA from liver samples measured by qPCR, b) Iron tissue content in liver per gram of wet tissue weight, c) Red blood cell count (RBC), d) Hemoglobin level (Hb), e) Hematocrit (HCT), f) Mean corpuscular volume (MCV), g) Red blood cell distribution width (RDW), h) Mean corpuscular hemoglobin (MCH), i) Number of erythroid cells at developmental stages I-V in the spleen determined by flow cytometry, j) Number of erythroid cells at developmental stages I-V 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. [Examples]

[0300] Antibodies targeting MTP-2 for the treatment of iron overload diseases are described herein. By immunizing transgenic mice that produce antibodies with a human variable domain and testing a wide variety of antibodies in a series of biologically relevant assays, we were able to obtain species cross-reactive MTP-2-specific monoclonal antibodies (mAbs) that serve as cross-reactive neutralizers of MTP-2 enzyme activity both in vitro and in vivo. We show that selected mAbs increase the level of hepcidin expression from hepatic cells after a single dose. The increase in hepcidin reduces serum iron and transferrin saturation by increasing the internal translocation and degradation of ferroportin. Hb, a model of beta-thalassemia. th3 / + In mice, a single dose of 10 mg / kg resulted in a 52% and 47% decrease in serum iron and transferrin saturation, respectively, at 2 weeks. Furthermore, with repeated administration, the inventors found that Hbb th3 / + We observed consistent iron restriction over multiple weeks in mice. These results suggest that such mAbs have the potential to treat iron overload in patients, reducing anemia and decreasing the need for transfusions and iron chelation. [Example 1]

[0301] Panel creation of anti-MTP-2 inhibitory antibodies Kymab transgenic mice producing antibodies with a human variable domain were immunized with MTP-2 using various 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. Antibodies were tested in protein-based and cell-based in vitro assays for binding to human and mouse MTP-2, as well as for their ability to inhibit the enzymatic activity of human and mouse MTP-2.

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

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

[0304] Next, the selected antibodies were screened in several functional assays to evaluate their ability to inhibit the enzymatic activity of purified human and mouse MTP-2 ECD in solution, as well as their ability to inhibit the enzymatic activity of human MTP-2 expressed in HEK293 cells, as assessed in enzyme assays containing chromogenic MTP-2 substrates.

[0305] We obtained a wide variety of antibodies, including 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. However, we selected the antibodies listed in Table G below as particularly interesting from the perspective of their potential for development as MTP-2 activity cross-reactivity inhibitors.

[0306] [Table 1] [Example 2]

[0307] antibody sequence Table S shows the sequences of the HCDR, LCDR, VH domain, and VL domain of antibodies NORI-001 to NORI-033, respectively. The complete IgG4PE heavy chain of each antibody is shown. The complete light chain of each antibody is also shown. Unless otherwise indicated in the context, "NORI-001" refers to the antibody having the VH and VL domains shown as NORI-001 in Table S. The composition of the antibody may also be shown; for example, "NORI-001 IgG" is IgG having the NORI-001 VH domain and the NORI-001 VL domain. "scFv" is an scFv that has the NORI-001 VH domain and the NORI-001 VL domain.

[0308] The antibody NORI-002 was found to have a high-risk free cysteine ​​liability in its VH domain. While this amino acid was thought to be replaceable through structural software analysis, mutation was introduced to mitigate all potential defects. Therefore, the C49C mutation was introduced into NORI-002, and the new antibody containing this mutation was designated NORI-003.

[0309] The antibody NORI-010 is a sequence that has been optimized by introducing the P124S mutation into the VH domain to improve stability and expression, thereby creating a new antibody NORI-011. [Example 3]

[0310] Inhibition of MTP-2 in a protein-based enzyme assay using MTP-2 ECD The antibodies were evaluated for their ability to inhibit serine protease cleavage of labeled MTP-2 substrates and generate detectable products in enzyme assays 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 human MTP-2 inhibition ranged from approximately 1.5 to 55 nM. The IC50 values ​​for mouse MTP-2 inhibition ranged from approximately 0.48 to 40 nM. Table D. Figure 3.

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

[0313] Antibody cross-reactivity was evaluated by comparing inhibition in assays using human MTP-2 ECD with inhibition in assays using mouse MTP-2. By plotting the inhibition percentages from the two assays against each other, it was observed that some antibodies were specific to human MTP-2, while others were specific to mouse MTP-2, and that varying degrees of inhibition existed for both human and mouse MTP-2. Figure 4.

[0314] Materials and methods for protein-based enzyme assays In the initial assay, positive and negative controls, i.e., aprotinin (Sigma-A3428) and non-MTP-2 conjugated huIgG4PE isotype antibodies, were serially diluted 1:3 from a starting concentration of 200 nM to a 2x final concentration of assay buffer (200 mM Tris-HCl and 1 mg / mL BSA, pH 9.0), respectively. In subsequent assays, the positive and negative controls used were 10 nM NORI-008 huIgG4PE and non-MTP-2 conjugated huIgG4PE isotype antibodies, respectively.

[0315] Titrated control and diluted antibodies (20 μl each) were plated onto 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 final concentrations of 0.5 or 2 mg / ml, respectively, to account for differences in enzyme activity. The plate was then covered and incubated at room temperature for 30 minutes. Next, 10 μl of the fluorescent MTP-2 peptide substrate Boc-Gln-Gly-Arg-AMC (Bachem AG - 4016429.0050) was added to each well at a final concentration of 50 μM in assay buffer. The enzymatic reaction was allowed to proceed at room temperature, and the fluorescence activity was read using a plate reader (Envision) at excitation wavelengths of 360 nm and emission wavelengths of 460 nm at 30 minutes, 1 hour, and 2 hours. When the substrate peptide is cleaved via the enzymatic activity of the MTP-2 enzyme, the 7-amide-4-methylcoumarin (AMC, MCA, or NHMec) moiety is released from the C-terminus. AMC is a fluorophore that is incorporated into the carboxypeptidase substrate at its C-terminus. Subsequently, excitation of the released coumarin occurs at 360–380 nm using a spectrofluorometer, and emission can be detected at 440–460 nm. Positive and negative controls were obtained as the average of 16 wells of aprotinin / NORI-008 IgG and huIgG4PE isotype at a final concentration of 200 nM, respectively. Inhibition values ​​were measured using graphpad. By inputting data into the software, a logarithmic curve was created, and IC50 values ​​were generated using nonlinear regression parameters and a logarithmic (inhibitory) versus response-variable slope (four parameters) equation.

[0316] For details on the preparation of the antigen reagent, please refer to Example 5. [Example 4]

[0317] Inhibition of MTP-2 in cell-based enzyme assays using MTP-2 expressed on the cell surface. The antibody was evaluated for its ability to inhibit serine protease cleavage of labeled MTP-2 substrates and generate detectable products in an enzyme assay using human MTP-2 expressed on the cell surface.

[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 on the cell surface, which is the site where cells express activated MTP-2 that undergoes self-activation and cleaves substrates. Inhibitors in this assay can act through various molecular mechanisms. For example, inhibitors that exhibit inhibition in this assay may bind to the MTP-2 enzyme precursor and inhibit its conversion to self-activated MTP-2 (Figure 2), thereby preventing the formation of the activated form of MTP-2 that would otherwise cleave substrates in this assay, and / or the inhibitors in this assay may bind to and inhibit the activated form of MTP-2.

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

[0321] Materials and methods for cell-based enzyme assays All antibodies for screening, as well as for positive and negative controls, were serially diluted 1:3 from a starting concentration of 200 nM in 2x final concentration Expi293 medium (A1435101) (serum-free medium supplemented with high glucose and GlutaMAX, pH 8). 12.5 μl of antibodies (protein A purified) and controls 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 to the 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 following day, fluorescence activity was read using a plate reader (Envision) at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. Positive and negative controls were obtained as the average of four wells of aprotinin and huIgG4PE isotype at a final concentration of 200 nM, respectively.

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

[0323] For details on the preparation of the antigen reagent, please refer to Example 5. [Example 5]

[0324] Manufacturing of antigen materials Generating constructs for protein expression To produce purified protein for use in the assays described herein, • Extracellular domain (ECD) aa77~855 of WT human matryptase-1 (uniprot sequence number Q9Y5Y6) • Wild-type (WT) human MTP-2 (uniprot sequence number Q8IU80) aa78~811 • WT mouse MTP-2 (uniprot sequence number Q9DBI0) aa80~811 • WT rat MTP-2 (NCBI sequence number XP006242057.1) aa80~811 • Cryptomolgus macaque MTP-2 (uniprot sequence number A0A2K5VAP0) aa73~800 The DNA sequence encoding was used.

[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 reader sequence, and then codon-optimized for mammalian expression before expression.

[0327] To generate the MTP-2 antigen, co-expression with untagged moHAI-2 ECD antigen (followed by purification of the His-tagged antigen via a nickel column) can significantly improve expression. For this purpose, the DNA encoding WT mouse HAI-2 (uniprot sequence number Q9WU03) aa28~197 was fused with an N-terminal immunoglobin reader sequence, codon-optimized for mammalian expression, and then expressed.

[0328] The DNA sequence was cloned into a 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 creation To screen for MTP-2-specific antibodies, we created stable cell lines expressing the relevant antigens.

[0330] The full-length DNA sequences encoding wild-type (WT) human MTP-2 (uniprot sequence number Q8IU80) amino acids (aa)1-811 and WT mouse MTP-2 (uniprot sequence number Q9DBI0) aa1-811, fused with the N-terminal eGFP and C-terminal flag tag (DYKDDDDK), respectively, were codon-optimized for mammalian expression. This process was repeated for untagged cynomolgus monkey MTP-2 (uniprot sequence number A0A2K5VAP0) aa1-800. The DNA sequences were cloned into expression vectors under the control of a CMV promoter adjacent to 3' and 5' piggyBac-specific terminal repeat sequences to facilitate stable integration into the cell 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 the creation of stable cell lines.

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

[0332] The full-length DNA sequences encoding WT human matryptase-1 (MTP-1) (uniprot SEQ ID NO: Q9Y5Y6) aa1~855 with C-terminal His tag fusion, WT human matryptase-3 (MTP-3) (uniprot SEQ ID NO: Q7RTY8) aa1~854 with C-terminal His tag fusion, and tagless WT mouse HAI-2 (uniprot SEQ ID NO: Q9WU03) aa1~252 were all codon-optimized for mammalian expression and cloned into the same expression vectors with the CMV promoter as before. The expression vectors for MTP-1 and MTP-3 contained a puromycin-selective cassette, and the expression vector for moHAI-2 contained a neomycin-selective cassette to facilitate the creation of dual-stable cell lines.

[0333] Production of stable transfectable Hepa1-6, CHO, and HEK293 cells expressing MTP-1, MTP-2, and MTP-3 antigens. To generate human embryonic kidney (HEK) 293 cell lines expressing WT human MTP-2 eGFP / Flag-tagged aa1-811, a CMV promoter expression plasmid was co-transfected into human embryonic kidney (HEK) 293 cells along with a plasmid encoding piggyBac transposase using FreeStyle Max transfection reagent (Invitrogen) according to the manufacturer's instructions.

[0334] To create Chinese hamster ovary (CHO) cell lines expressing WT human MTP-2 eGFP / Flag-tagged aa1-811, a CMV promoter expression plasmid was co-transfected into Chinese hamster ovary (CHO) cells along with a plasmid encoding piggyBac transposase using FreeStyle Max transfection reagent (Invitrogen) according to the manufacturer's instructions.

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

[0336] To create 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 co-transfected into HEK293 cell lines using FreeStyle Max transfection reagent (Invitrogen) according to the manufacturer's instructions, along with a WT moHAI-2 expression plasmid and a plasmid encoding piggyBac transposase.

[0337] 24 hours after transfection, add puromycin (2.5 μg / mL) or G418 (1 mg / mL), or puromycin (2.5 μg / mL) and G4 to the culture medium. Both 18 (1 mg / mL) were supplemented, and the cells were grown for at least two weeks to select stable cell lines. 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 monoclonal cell lines for the highest expression. This was repeated for 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 evaluated by flow cytometry using an anti-flag APC conjugate antibody (Biolegend - 637308). Expression of his-tagged human MTP-1, human MTP-3, and human and mouse MTP-2 constructs was evaluated by flow cytometry using an unlabeled anti-His primary antibody (Abcam - ab18184) followed by a goat anti-mouse 647 conjugate secondary antibody (Citeab - 115-605-071). After selection, stable untagged WT human MTP-2 and WT mouse MTP-2 expressing HEK293 cells, as well as stable untagged WT human MTP-2 and WT mouse MTP-2 expressing Hepa1-6 cells, were FACS-selected for high expression. Cellular expression of untagged human, mouse, and cynomolgus monkey MTP-2 constructs, including human and mouse variants and human variant constructs, was evaluated by flow cytometry using an APC-conjugated anti-MTP-2 antibody.

[0338] HEK293 and Hepa1-6 complete media consisted of Dulbecco's Modified Eagle 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 a suspension containing CHO-S complete medium consisting of CD-CHO medium supplemented with 8 mM Glutamax (Gibco). The CHO-3E7 cells used for expression here included the pTT5 vector system and CHO EBNA 1 cells available from the National Research Council of Canada, but other CHO cell lines may be used. [Example 6]

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

[0340] Affinity (K) for the tested antibody D The range was approximately 0.012 nM to 8.5 nM. (Table K)

[0341] [Table 3]

[0342] Furthermore, binding was evaluated using the SPR method described above with "headless" huMTP-2 ECD 78~576aa instead of complete ECD. Headless ECD corresponds to MTP-2 ECD that lacks a serine protease domain. The amino acid sequence of the "headless" huMTP-2 ECD 78~576aa protein corresponds to the human matryptase-2 mask 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 of 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 were, respectively, represented in Table S as human matryptase-2 ECD protein his tag, mouse matryptase-2 ECD protein his tag, cynomolgus monkey matryptase-2 ECD protein his tag, rat matryptase-2 ECD protein his tag, and human matryptase-2 mask ECD protein his tag. The results were given as follows. No binding to the human matryptase-2 mask ECD protein his tag was detected for any of these four antibodies, which indicates that the binding is not to the his tag and that the epitopes of all antibodies reside in the serine protease domain. The true KD value of NORI-008 could not be accurately measured for both human MTP-2 and mouse MTP-2 due to the extremely slow dissociation rate. [Example 7]

[0345] Binding to the MTP-2 serine protease domain SPR analysis performed on the antibody panel in Example 6 showed no detection of binding to the truncated "headless" huMTP-2 protein lacking the serine protease domain. This suggests that these antibodies bind to the serine protease domain of MTP-2. Such antibodies are expected to mechanistically block MTP-2 by blocking or deforming the serine protease domain active site, thereby preventing substrate cleavage.

[0346] HTRF binding assays of NORI-003, NORI-006, NORI-008, and NORI-011 were performed against this headless protein, and no detectable binding was found (Figure 6). However, binding was detected against all human, mouse, and cynomolgus monkey MTP-2 proteins that do not have C-terminal shortening (Figure 7). This also suggests that the binding epitopes of these inhibitory antibodies are located in the serine protease domain. Another antibody, NORI-036, bound to both headless ECD and complete ECD, indicating that NORI-036 recognized the MTP-2 binding site outside the serine protease domain. [Example 8]

[0347] Competition with aprotinin Aprotinin is a panserine protease inhibitor and is 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 site and competed with aprotinin for binding to the human MTP-2 protein. For NORI-003 and NORI-006, this competition was weak despite high 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 aprotinin binding site. This was confirmed by the lack of competition with aprotinin for moMTP-2 in NORI-003 and NORI-006, despite their IC50 values ​​being comparable to those of NORI-011 in the enzyme assay. NORI-008 exhibited very low Kd and IC50 values ​​in SPR and aprotinin competition assays, respectively, suggesting that its binding site was identical to or strictly overlapped with that of aprotinin. Figure 8. Table L.

[0348] [Table 5]

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

[0350] Progress in in vivo research MTP-2 has been recognized as a target for addressing iron-overload anemia by increasing hepcidin levels through various models and concepts. The absence or blockade of MTP-2 activity can increase hepcidin levels and thus reduce iron overload, as demonstrated in human genetics, preclinical models, and clinical interventions. For example, Tmprss6 knockout mice are viable but characterized by iron restriction, resulting in high hepcidin levels and a transient, so-called "masked" phenotype that causes body hair loss but not scalp hair loss. Crossing these mice with beta-thalassemia mice with hemizygous beta-globin chain loss reveals the same phenotype seen in these hybrids. Increased hepcidin levels can improve red blood cell count and hemoglobin levels (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 that cannot be cured by administering more iron (iron-refractory iron deficiency anemia - IRIDA) (Lenoir et al., Blood 117:647~650 2011; Nai et al., 2012, cited above).

[0351] Inhibition of MTP-2 activity suggests that antibodies can increase hepcidin expression, thus potentially preventing iron overload, a major cause of β-thalassemia and death in β-thalassemia. Therefore, antibodies exhibiting such activity in vivo may improve anemia in β-thalassemia and could be useful therapeutic agents for treating patients with iron overload or at risk of iron overload.

[0352] In vivo evaluation included assessing increased hepcidin gene mRNA transcription and decreased serum iron levels from hepatocytes in wild-type mice. Readouts of this evaluation were obtained within 24 hours of administration, enabling rapid confirmation of antibody activity.

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

[0354] Antibodies selected for in vivo testing were chosen based on their concentration-dependent inhibition of enzyme activity against human and mouse MTP-2 protein and MTP-2 expressed on the cell surface. Antibodies were excluded if they were considered to have potential drawbacks regarding expression and / or purification yield, posing a risk to development feasibility, or if complete enzyme inhibition was not observed in vitro against either human or mouse ECD protein, or if the generated IC50 value was considered very low.

[0355] Two antibodies that showed weak inhibition in the enzyme assay were also tested in vivo. Both exhibited very poor in vivo function, 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, this may not necessarily be a sufficient condition for achieving an in vivo effect. For some antibodies, we observed in vivo performance that was potent in the short term but not maintained over time. Other factors, such as pharmacokinetics or anti-drug antibodies produced by mice, may influence the 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 Iron quantification was performed using the QuantiChrom® iron assay kit (Bioassay System, DIFE-250) to generate the data shown in Figures 13, 14, 15, 16, 17, and 18.

[0357] In short, iron standards were prepared according to the kit protocol. Then, 25 μl of the 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 using a microplate spectrophotometer (reading A). Next, 10 μl of reagent B was 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 using a plate reader (reading B). For all wells, the increase in absorbance was calculated by subtracting reading A from reading B. The standard curve was then plotted, and the unknown value of sample iron was 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 a direct iron assay (Ferene) kit, following the protocol provided in the kit.

[0359] In short, 25 μl of serum from a standard or research animal was incubated in a 96-well plate with 125 μl of solution R1 and 25 μl of solution R2, and the plate was then read using an Envision microplate reader according to the 600 nm CLF protocol (absorbance A1). 2.5 μl of chromogen was added. After incubation at room temperature for 20 minutes, the plate was read according to the same Envision protocol (absorbance A2). The results were:

number

[0360] Any value calculated to be less than 0 will be reported as 0.

[0361] Calculation of transferrin saturation (TSAT) TSAT analysis was performed using an iron-fixed latency measurement kit according to the protocol provided in the kit. Briefly, 25 μl of serum from a standard or study 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 using an Envision microplate reader based on the 600 nm CLF protocol (absorbance A1). 25 μl of serum from a standard or study animal was incubated with 125 μL of standard solution (R1:R2 ratio of 50:1) at room temperature for 5 minutes. The plate was then read using the same Envision protocol as before (absorbance A2). The results were:

number

[0362] Any value calculated to be less than 0 will be reported as 0.

[0363] Analysis of serum antibody levels Serum antibody concentrations were confirmed using the following assay: 50 μL / well of mouse anti-human IgG4 Fc (2 μg / mL in PBS) was coated overnight at 4°C in a 96-well plate. The plates were washed three times using a plate washer with 300 μL / well of PBD-T (PBS and 0.1% Tween). The plates were blocked at room temperature for 1 hour with PBS supplemented with 150 μL / well of 1% BSA. Samples were diluted (using stored mouse serum), and QC and standard curves (10 levels (7.81–2000 ng / ml)) were prepared. The plates were then washed three times using a plate washer with 300 μL / well of PBD-T (PBS and 0.1% Tween). 50 μL of standard curve, sample, or QC per well was added to the assay plate, and the assay plate was incubated at room temperature for 1 hour with shaking at 300 RPM. The plates were then washed three times using a plate washer with 300 μL / well of PBD-T (PBS and 0.1% Tween). 50 μL / well of HRP-conjugated mouse anti-human kappa, diluted 1 / 12000, was added to PBS with 1% BSA, and the plate was 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 and 0.1% Tween) using a plate washer. 100 μL of TMB substrate was added to each well. The plates were incubated at RT for 10 minutes in the dark. Then, 100 μL / well of stop solution (1M sulfuric acid) was added. The optical density of each well was measured using a microplate reader set to 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 coefficient of 1 / y for the sample concentration standard 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 protocol. Frozen samples were thawed on wet ice. 600 μl of Buffer RLT Plus was then added to the samples. The samples were then homogenized using a plastic pestle. The samples were then tritium-labeled using a 1 ml syringe and a 20 G needle. The samples were then centrifuged, and the supernatant was placed in a 2 ml Eppendorf gDNA-removing spin column, with the pellet discarded. The spin column and tube were centrifuged, the flow-through was retained, and the column was discarded. 600 μl of 70% ethanol was then added to the flow-through. 700 μl of the sample was then placed on an RNeasy Spin Column in a 2 ml recovery tube and centrifuged. The flow-through was discarded. The spin column was then returned to the 2 ml recovery 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 a 2 ml recovery tube, 500 μl of Buffer RPE was added to the spin column, and the spin column was centrifuged. The flow-through was discarded, and the column was washed with Buffer RPE as described above. The column was centrifuged at 8000 × g for 15 seconds. The spin column was placed in a new 2 ml recovery tube and centrifuged at a maximum of 1 minute to dry the membrane. The spin column was placed in a new 1.5 ml recovery 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 level in the flow-through was estimated using nanodrop (see below), and the solution was then stored at -20°C or -80°C.

[0365] qPCR analysis After mRNA extraction, mRNA from the liver of each mouse was quantified by nanodrop and all were normalized to 5 ng / μl. Briefly, the transcript level of mouse hepcidin (hamp) mRNA was measured by qRT-PCR and normalized to the mouse hypoxanthine-guanine phosphoribosyltransferase (HPRT) mRNA housekeeping gene. Then, 5 μl of mRNA extract (25 ng total) was mixed with 10 μl of QuantiTect probe RT-PCR kit and 20X hamp FAM probe mix. 1 μl of kus, 1 μl of 20X HPRT VIC probe mix, 40X taqman 0.5 μl of RT enzyme mix was mixed with 2.5 μl of RNA-free H2O 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 reverse transcription step of 48°C for 15 minutes, followed by an activation step of 95°C for 10 minutes, and then 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. The -ΔCt value was then calculated by subtracting the Hamp Ct value from the HPRT. [Example 11]

[0366] Evaluation of single-dose IgG / lambda anti-MTP2 antibodies over 24 hours in normal mice for reducing serum iron and transferrin saturation. NORI-009, NORI-010, NORI-012, and NORI-034 were included as full human IgG4λ mAbs in the first evaluation of healthy mice. One intraperitoneal injection antibody was administered to 9-week-old C57BL / 6 male mice at a dose of 10 mg / kg (150 μl / mice) and 5 mice per group. Mice were sacrificed 24 hours after injection. As a positive control for hepcidin induction, three 9-week-old C57BL / 6 male mice were injected with LPS (1 μg per g of body weight) and sacrificed 4 hours later. Because LPS (lipopolysaccharide derived from E. coli) mimics bacterial infection, mice respond by inducing hepcidin expression and decreasing serum iron. LPS serves as a positive control for drugs known to induce an acute inflammatory response and, based on the inflammatory response, induce a maximum hepcidin increase. Real-time PCR of 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 and transferrin saturation was calculated from all mice.

[0367] NORI-034 did not increase hepcidin expression beyond the levels observed in isotype-controlled mice. On the other hand, NORI-009, NORI-010, and NORI-012 all increased hepcidin expression to at least 1 ct value above the mean of the group treated with isotype controls 24 hours after administration, and were equal to the hepcidin expression induced by LPS-positive controls 4 hours after administration. As a result, serum iron content in mice treated with these antibodies decreased to a mean concentration of less than 40 μg / dl. Figure 9.

[0368] This experiment demonstrated a biological link between MTP-2 and the BMP / SMAD / hepcidin pathway, and for the first time showed that antibody-guided inhibition can reduce serum iron levels in normal mice. Since NORI-034 was considered inactive in vivo, this antibody was not further explored. [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 interesting to determine the effects of this antibody observed over time. One intraperitoneal injection antibody was administered to C57BL / 6 male mice at a dose of 10 mg / kg (150 μl / mice) and 5 mice per group. The groups consisted of 5 mice sacrificed at 24 hours, 72 hours, 1 week, and 2 weeks. The analysis was the same as in Example 11, but also included hematocrit (HCT), mean corpuscular hemoglobin (MCH), and erythrocyte distribution width (RDW), a measure of cell diameter related to erythrocyte volume.

[0370] Administration of 10 mg / kg of antibody maintained hepcidin elevation for two weeks, during which time serum iron and TSAT levels decreased. MCV levels decreased due to consistent iron restriction. Figure 10.

[0371] With 3 mg / kg antibody administration, the effect on hepcidin elevation was lost by 2 weeks due to the lower dose. Nevertheless, serum iron and TSAT remained decreased over 2 weeks, and MCV was also reduced, similar to the case with the 10 mg / kg dose. Figure 11. [Example 13]

[0372] Evaluation of two IgG / kappa anti-MTP2 antibodies and two IgG / lambda anti-MTP-2 antibodies over 24 hours regarding their ability to reduce serum iron and transferrin saturation. NORI-008, composed of mouse IgG1 (moIgG1), 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 composed of moIgG1 and huIgG4PE, respectively. The latter control antibodies were cross-reactive binders for MTP-2, but in vitro they did not inhibit the enzymatic activity of either human or mouse ECD protein.

[0373] Nine-week-old C57BL / 6 male mice were administered one intraperitoneal injection antibody at a dose of 10 mg / kg (150 μl / mice) in groups of 3-5 mice. Mice were sacrificed 24 hours 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 was determined and transferrin saturation was calculated from all mice.

[0374] Here, the inventors found that NORI-008, which has a very strong affinity for human and mouse MTP-2, is also active in vivo. In comparison with NORI-010, it was found to be active in that it increased hepcidin mRNA expression and decreased 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 for MTP-2, but they have no effect on hepcidin expression or serum iron in vivo. This demonstrates that the in vitro screening strategy succeeded in producing 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 injections of 10, 3, and 1 mg / kg into normal mice. This experiment demonstrates the dose-time relationship after intravenous injection of the anti-MTP-2 antibody NORI-008, as measured by changes in serum iron concentration.

[0377] Male C57BL6J mice weighing 22-28g (n=5 / group) were administered a single intravenous 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 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), and each group was selected with a different dose. Blood samples were collected from one group of animals treated with each dose on days 1, 7, and 14, and serum iron levels were checked to evaluate the efficacy of NORI-008.

[0378] Next, samples from all doses and time points were analyzed for hepcidin expression in the liver and antibody levels in serum on day 7 (PK analysis). Antibodies were present at all time points in the 2 μg group. Even then, the levels were unmeasurable, and 24 hours after a 20 μg dose, only 3 out of 5 animals had measurable levels.

[0379] At a 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 a 20 μg dose, the effect lasted for at least one day, after which it returned to normal at one week. At a 2 μg dose, no effect was observed. The duration of the effect correlated with the PK value of the antibody concentration. That is, when the antibody level fell below the critical concentration, the effect on MTP-2 inhibition was lost, and hepcidin expression and serum iron returned to normal. Figure 13. [Example 15]

[0380] In vivo evaluation at 1 week in normal mice of six IgG4 / kappa anti-MTP2 antibodies for reducing serum iron and transferrin saturation. This study evaluated the ability of six fully human IgG4 anti-MTP-2 antibodies to lower serum iron levels after intravenous injection (IP).

[0381] Male C57BL6J mice weighing 25-32g (n=3-5 per group) were administered a single 10 mg / kg intravenous injection of a fully human IgG4 anti-MTP-2 antibody. Eight groups were included in the study, and each animal received either one of the seven fully human IgG4PE anti-MTP-2 antibodies (NORI-005, NORI-004, NORI-002, NORI-001, NORI-007, NORI-006, and NORi-010) or a single 10 mg / kg intravenous injection of a human IgG4 isotype control antibody. NORI-010 was included as a positive control and benchmark anti-MTP-2 antibody for comparison with the remaining antibodies used in this experiment. All groups, except for NORI-005 (n=3) and NORI-002 (n=4), contained five animals. Animal blood was collected on day 7, serum iron concentration was checked, and iron parameters, hamp mRNA, and IgG levels of anti-MTP-2 antibody were evaluated.

[0382] All tested antibodies showed increased hepcidin mRNA expression, and therefore a corresponding decrease in serum iron levels. Next, serum antibody levels in samples from all tested groups were analyzed on day 7 (PK analysis), showing the varying levels of antibody exposure at this time and dose. Figure 14. [Example 16]

[0383] In vivo evaluation at 1 week of the effects of two IgG / kappa anti-MTP2 antibodies with both huIgG4 and moIgG1 skeletons and one IgG / lambda anti-MTP2 antibody (10 mg / kg IP dose) on reducing serum iron levels. This study evaluated the ability of three anti-MTP-2 antibodies, either fully human IgG4 or mouse IgG1, to lower serum iron levels after subcutaneous injection.

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

[0385] Figure 15. [Example 17]

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

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

[0388] Animal blood was collected on day 7, and serum iron concentration and IgG levels were checked. Figure 16.

[0389] The results of this experiment show that serum antibody concentrations were comparable at one week for both the iP and scP administration methods. Consequently, serum iron levels were also similar for both administration methods and were reduced by MTP-2 inhibition, as previously observed. [Example 18]

[0390] In vivo evaluation of dose / time response after single intravenous 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 260-320g (n=3 / group) were administered a single intravenous injection of 3 or 10 mg / kg of fully human IgG4 anti-MTP-2 antibodies NORI-008 and NORI-010. A 10 mg / kg hIgG4 isotype control antibody (indicated as "isotype") was administered via intravenous injection as a negative control.

[0392] Animal blood was collected on days 1, 3, 7, 9, 14, and 21. Serum iron and antibody concentrations were measured at all time points.

[0393] NORI-010 produced a pharmacological effect at 10 mg / kg, reducing serum iron to 9 days before returning to normal, but the effect lasted only 24 hours at 3 mg / kg. NORI-008 produced an effect at 10 mg / kg, reducing serum iron to 9 days before returning to normal, but the effect lasted only 72 hours at 3 mg / kg. 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 single sc injection of anti-MTP-2 antibodies NORI-003, NORI-006, NORI-008, and NORI-010 in normal rats. In vivo evaluations of the dose / time response after a single sc injection of anti-MTP-2 antibody were performed in normal rats.

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

[0396] Animal blood was collected on days 1, 3, 7, 9, 14, and 21. Serum iron and antibody concentrations were measured at all time points.

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

[0398] PK analysis in this study reveals significant differences in Cmax among the four antibodies, with NORI-003 and NORI-006 having significantly higher serum antibody concentrations on day 3 compared to NORI-008 and NORI-010. Consequently, 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. Therefore, their effect on suppressing serum iron is sustained. Figure 18. [Example 20]

[0399] In vivo evaluation of beta-thalassemia in a mouse model after a single IP injection of NORI-010. The selected antibody NORI-010 was evaluated in a heterozygous mouse model of beta-thalassemia intermedia (Hbbth3 / +) with heterozygous deletions of the b1 and b2 globin genes. Hbbth3 / + mice exhibit a complex phenotype that worsens with age, including Hb levels between 7–9 g / dL, abnormal erythrocyte morphology, increased reticulocyte count, ineffective and extramyeloid erythropoiesis, hepatosplenomegaly, and iron overload in the liver and spleen. According to the activity of NORI-010 observed in Examples 18–20, 10 mg / kg of this antibody was administered by ip injection, and the animals were sacrificed 2 weeks after this single dose (n=3 per time point). In addition, hepcidin mRNA was analyzed in one group of animals 24 hours later. A human IgG4 isotype control antibody was used as a negative control at the same dose.

[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 (for 2 weeks according to the method in Example 10) - Calculated transferrin saturation (after 2 weeks according to the method of Example 10) - Mean corpuscular volume (2 weeks)

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

[0402] Evaluation of 8-week repeated administration of NORI-011-M in a mouse model of beta-thalassemia, with and without concurrent erythropoietin treatment. The purpose of this study was to investigate the effects on hematological parameters after a longer treatment period than in Example 20. For this purpose, Hbbth3 / + mice (n=5 / group) were intraperitoneally injected with NORI-011-M antibody at a dose of 10 mg / kg once a week for 8 weeks. For this longer-term repeated-dose experiment, the NORI-011 isotype was reconstituted with respect to the mouse constant region (mouse IgG1 and mouse lambda constant region) to avoid or suppress immunogenicity and anti-MTP2 antibody production in mice. In connection with this reconstitution, a minor modification was also made to the C-terminal sequence of the NORI-011 VH domain, and the modified antibody was newly named 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 attempted to investigate the effects of co-administration of erythropoietin (epo), which is known to improve some important parameters but has adverse effects on spleen size due to iron overload and excessive cell death. The favorable effects of this combination had been confirmed in experiments using Hbbth3 / + mice, where epo was supplied by overexpressing recombinant fibroblasts and matryptase-2 activity was reduced by using Tmprss6 antisense oligonucleotide treatment (Paper No. 164, 60th Annual Meeting of the American Society of Hematology, 2018). In this study, clinical-grade epo (darbepoetin alfa) was administered once a week at 30 μg / kg co-administered with NORI-011-M to one group of the study. As a control, epo was also administered co-administered with an isotype control antibody to demonstrate the effect of epo alone on readout in this study. Figure 20 shows the results for individual hematological parameters and hepcidin mRNA levels in liver samples.

[0404] The results also indicate that, in this case as well, hepcidin levels were consistently maintained at elevated levels throughout the 8-week experiment (a). Therefore, as seen in the above examples, this is expected to result in iron restriction, as well as subsequent reductions in serum iron and hepatic iron content (b) and MCV (f). Red blood cell count and hemoglobin did not substantially increase with NORI-011-M alone. However, red blood cell maturation improved in the spleen, with a much higher percentage of stage V (mature) cells than in any other group (i). Increased spleen weight was also suppressed by about half with NORI-011-M alone, indicating that the restriction of iron supply caused by the blocking agent of NORI-011-M has a normalizing effect on red blood cell maturation (k).

[0405] The use of epo (in combination with an isotype control) has a favorable effect on red blood cell count (c) and hemoglobin (d), as expected, as a stimulant of erythropoiesis. However, as has been previously known, the increased erythropoiesis with epo use, in the context of beta-globin synthesis deficiency, leads to increased cell death and associated spleen size (index) (k), and does not improve maturation to functional and high-quality red blood cells (i). As expected, epo failed to increase hepcidin levels and therefore had no effect on hepatic iron levels (c).

[0406] The combination therapy of NORI-011-M and epo yielded a favorable combination of effects. While there were slightly reduced effects on red blood cell count (c) and hemoglobin (d) compared to epo alone, overall, it was represented by a more balanced therapeutic effect compared to epo alone, including a sustained reduction in hepatic iron overload (b) and a decrease in splenomegaly (k). These synergistic effects in clinical practice are likely to be optimized by optimizing the treatment ratio of matryptase inhibition to epo stimulation. [Example 22]

[0407] Evaluation of 8-week repeated doses of NORI-011-M in a mouse model of beta-thalassemia, with and without concurrent treatment with ActRIIB-Fc fusion protein. This study demonstrates that co-treatment with NORI-11-M and activin receptor IIB Fc fusion protein can achieve beneficial therapeutic effects compared to treatment with NORI-11-M alone or ActRIIB-Fc alone. This synergistic effect is attributed to ActRIIB This may reflect the different mechanisms of action of these two drugs: -Fc promotes the maturation of erythrocyte precursors during erythrocyte production, while NORI-11-M causes slowing of erythrocyte production, leading to iron restriction and normalization, and thus more efficient production of more mature erythrocytes.

[0408] The work in this study was carried out over 8 weeks with multiple doses of both drugs using the methods and procedures outlined in Example 21 above. As before, Hbbth3 / + mice (n=5 / group) were intraperitoneally injected with NORI-011-M antibody 10 mg / kg once a week for 8 weeks. ActRIIB-Fc 10 mg / kg was injected intraperitoneally twice a week for 8 weeks. The number of times ActRIIb-Fc was administered, twice as many times as the antibody, reflected previously published administration 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 β-thalassemia, Blood, 2014;123(25):3864~3872, doi:10.1182 / blood-2013-06-511238, and for ActRIIA-Fc, Dussiot et al., 2014).

[0409] The ActRIIB-Fc construct used in this example was expressed in suspension CHO cells and fused to a mouse IgG2a-Fc domain (uniprot - P01863, residues 99-330) via a short 3x glycine linker, fused to a modified human ActRIIB extracellular domain (uniprot - ) with an L79D modification, purified via the Fc domain. Q13705, residues 26-131)

[0410] The 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 also show that in this case, hepcidin levels were consistently maintained at elevated levels over the 8-week course using NORI-11-M (Figure 21a), demonstrating that NORI-11-M has the effect of increasing hepcidin expression levels, thereby causing a decrease in hepatic iron levels (Figure 21b), a decrease in serum iron, and a reduction in mean corpuscular volume (MCV) (Figure 21f). With treatment using ActRIIB-Fc alone, no increase in hepcidin (hamp) mRNA was observed, and concurrent treatment resulted in the same outcomes as when using NORI-11-M alone. Therefore, despite a greater number of doses, ActRIIB-Fc was unable to directly increase hepcidin expression or, consequently, reduce hepatic iron levels.

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

[0413] All treatment groups improved the "quality" of the red blood cells produced, as reflected in a reduction (normalization) of the red blood cell distribution width (RDW) (Figure 21g). Thus, the uniformity of red blood cells improved. With NORI-011-M alone, the red blood cell count increased slightly (Figure 21c), but neither hemoglobin (Figure 21d) nor hematocrit (Figure 21e) increased. ActRIIB-Fc had a greater effect on all of these parameters. The combination did not result in any further increase beyond that of ActRIIB-Fc alone, indicating that the maturation effect on red blood cell production was driven more by ActRIIB-Fc than by NORI-11-M. However, it is noteworthy that NORI-11-M did not counteract the effect of ActRIIB-Fc. Red blood cell counts recovered to wild-type levels, and both hematocrit and hemoglobin levels significantly improved with treatment using ActRIIB-Fc alone or in combination with NORI-11-M. This is significant because it indicates that the iron reduction caused by NORI-11-M does not negate the beneficial effect on erythrocyte production produced by ActRIIB-Fc. Therefore, overall, the benefits of both treatments can still be achieved, even when used in combination, making it possible to both reduce iron excess and increase erythrocyte production / maturation.

[0414] Furthermore, when the two drugs are used together, they actually provide greater overall benefits through their therapeutic efficacy, which surpasses that of using them individually, as demonstrated by their effectiveness in reducing splenomegaly, for example.

[0415] Red blood cell maturation improved in the spleen, with a much higher percentage of stage V (mature) cells than in any other group (Figure 21i). Increased spleen weight was suppressed in both treatments compared to the untreated control, indicating that even the limitation of iron supply caused by NORI-011-M had a normalizing effect on the efficiency of red blood cell maturation (Figure 21k).

[0416] In Example 21, the inventors reported that the use of EPO had a favorable effect on red blood cell count (Figure 20c) and total hemoglobin (Figure 20d), as expected for a stimulant 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 betaglobin synthesis deficiency, also leads to increased cell death and apoptosis, as well as a further increase in associated spleen size and weight, so-called splenomegaly (Figure 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 (Figure 21k). Furthermore, a significant reduction in spleen weight was actually observed, although not to the level of healthy animals. The combination treatment of NORI-011-M and ActRIIB-Fc was considered far 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 containing them, and their coding nucleic acids constitute 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. A binder polypeptide that binds to human MTP-2, comprising a heavy chain variable (VH) domain containing heavy chain complementarity-determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and a light chain variable (VL) domain containing light chain complementarity-determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, (a) HCDR1 contains the amino acid sequence of SEQ ID NO: 48, HCDR2 contains the amino acid sequence of SEQ ID NO: 49, HCDR3 contains the amino acid sequence of SEQ ID NO: 80, LCDR1 contains the amino acid sequence of SEQ ID NO: 98, LCDR2 contains the amino acid sequence of SEQ ID NO: 59, LCDR3 contains the amino acid sequence of SEQ ID NO: 88, or (b) HCDR1 contains the amino acid sequence of SEQ ID NO: 118, HCDR2 contains the amino acid sequence of SEQ ID NO: 119, HCDR3 contains the amino acid sequence of SEQ ID NO: 120, LCDR1 contains the amino acid sequence of SEQ ID NO: 128, LCDR2 contains the amino acid sequence of SEQ ID NO: 59, LCDR3 contains the amino acid sequence of SEQ ID NO: 129, or (c) A binder polypeptide in which HCDR1 contains the amino acid sequence of SEQ ID NO: 155, HCDR2 contains the amino acid sequence of SEQ ID NO: 137, HCDR3 contains the amino acid sequence of SEQ ID NO: 156, LCDR1 contains the amino acid sequence of SEQ ID NO: 164, LCDR2 contains the amino acid sequence of SEQ ID NO: 147, and LCDR3 contains the amino acid sequence of SEQ ID NO:

148.

2. The binder polypeptide according to claim 1, wherein HCDR1 consists of the amino acid sequence of SEQ ID NO: 48, HCDR2 consists of the amino acid sequence of SEQ ID NO: 49, HCDR3 consists of the amino acid sequence of SEQ ID NO: 80, LCDR1 consists of the amino acid sequence of SEQ ID NO: 98, LCDR2 consists of the amino acid sequence of SEQ ID NO: 59, and LCDR3 consists of the amino acid sequence of SEQ ID NO:

88.

3. The binder polypeptide according to claim 2, wherein the VH domain comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 94, and the VL domain comprises an amino acid sequence having at least 90% identity with SEQ ID NO:

100.

4. The binder polypeptide according to claim 2 or 3, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 94 and the VL domain comprises the amino acid sequence of SEQ ID NO:

100.

5. The binder polypeptide according to any one of claims 2 to 4, wherein the VH domain consists of the amino acid sequence of SEQ ID NO: 94 and the VL domain consists of the amino acid sequence of SEQ ID NO:

100.

6. A binder polypeptide according to any one of claims 2 to 5, comprising an antibody constant region.

7. A binder polypeptide according to any one of claims 2 to 6, comprising an IgG antibody, preferably comprising a YTE mutation.

8. A binder polypeptide according to any one of claims 2 to 7, comprising a human IgG4PE constant region.

9. A binder polypeptide according to any one of claims 2 to 8, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 96 and a light chain containing the amino acid sequence of SEQ ID NO:

102.

10. A binder polypeptide according to any one of claims 1 to 9, comprising a heavy chain consisting of the amino acid sequence of SEQ ID NO: 96 and a light chain consisting of the amino acid sequence of SEQ ID NO:

102.

11. The binder polypeptide according to claim 1, wherein HCDR1 consists of the amino acid sequence of SEQ ID NO: 118, HCDR2 consists of the amino acid sequence of SEQ ID NO: 119, HCDR3 consists of the amino acid sequence of SEQ ID NO: 120, LCDR1 consists of the amino acid sequence of SEQ ID NO: 128, LCDR2 consists of the amino acid sequence of SEQ ID NO: 59, and LCDR3 consists of the amino acid sequence of SEQ ID NO:

129.

12. The binder polypeptide according to claim 11, wherein the VH domain comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 124, and the VL domain comprises an amino acid sequence having at least 90% identity with SEQ ID NO:

132.

13. The binder polypeptide according to claim 11 or 12, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 124 and the VL domain comprises the amino acid sequence of SEQ ID NO:

132.

14. The binder polypeptide according to any one of claims 11 to 13, wherein the VH domain consists of the amino acid sequence of SEQ ID NO: 124 and the VL domain consists of the amino acid sequence of SEQ ID NO:

132.

15. A binder polypeptide according to any one of claims 11 to 14, comprising an antibody constant region.

16. A binder polypeptide according to any one of claims 11 to 15, comprising an IgG antibody, preferably comprising a YTE mutation.

17. A binder polypeptide according to any one of claims 11 to 16, comprising a human IgG4PE constant region.

18. A binder polypeptide according to any one of claims 11 to 17, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 126 and a light chain containing the amino acid sequence of SEQ ID NO:

134.

19. A binder polypeptide according to any one of claims 1 to 18, comprising a heavy chain consisting of the amino acid sequence of SEQ ID NO: 126 and a light chain consisting of the amino acid sequence of SEQ ID NO:

134.

20. The binder polypeptide according to claim 1, wherein HCDR1 consists of the amino acid sequence of SEQ ID NO: 155, HCDR2 consists of the amino acid sequence of SEQ ID NO: 137, HCDR3 consists of the amino acid sequence of SEQ ID NO: 156, LCDR1 consists of the amino acid sequence of SEQ ID NO: 164, LCDR2 consists of the amino acid sequence of SEQ ID NO: 147, and LCDR3 consists of the amino acid sequence of SEQ ID NO:

148.

21. The binder polypeptide according to claim 20, wherein the VH domain comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 170, and the VL domain comprises an amino acid sequence having at least 90% identity with SEQ ID NO:

166.

22. The binder polypeptide according to claim 20 or 21, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 170 and the VL domain comprises the amino acid sequence of SEQ ID NO:

166.

23. The binder polypeptide according to any one of claims 20 to 22, wherein the VH domain consists of the amino acid sequence of SEQ ID NO: 170 and the VL domain consists of the amino acid sequence of SEQ ID NO:

166.

24. A binder polypeptide according to any one of claims 20 to 23, comprising an antibody constant region.

25. A binder polypeptide according to any one of claims 20 to 24, comprising an IgG antibody, preferably comprising a YTE mutation.

26. A binder polypeptide according to any one of claims 20 to 25, comprising a human IgG4PE constant region.

27. A binder polypeptide according to any one of claims 20 to 26, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 172 and a light chain containing the amino acid sequence of SEQ ID NO:

168.

28. A binder polypeptide according to any one of claims 20 to 27, comprising a heavy chain consisting of the amino acid sequence of SEQ ID NO: 172 and a light chain consisting of the amino acid sequence of SEQ ID NO:

168.

29. A nucleic acid encoding a binder polypeptide according to any one of claims 1 to 28.

30. An in vitro host cell comprising the nucleic acid described in claim 29.

31. (i) a binder polypeptide according to any one of claims 1 to 28, and (ii) a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient.

32. The composition according to claim 31 for reducing the absorption of dietary iron in patients.

33. The composition according to claim 31 for treating iron overload in a patient.

34. The composition according to claim 31 for increasing the expression of hepcidin from hepatocytes in a patient.

35. The composition according to claim 31 for reducing anemia caused by iron overload in patients.

36. The composition according to claim 31 for reducing serum iron concentration in a patient.

37. The composition according to claim 31 for reducing the iron saturation of transferrin in a patient.

38. The composition according to any one of claims 32 to 37, wherein the patient has beta-thalassemia, myelodysplastic syndrome, polycythemia vera, hemochromatosis, or refractory anemia with ring sideroblasts (RARS).

39. (i) A binder polypeptide according to any one of claims 1 to 28, and (ii) A composition comprising an antagonist of a TGFβ superfamily ligand.

40. The composition according to claim 39, wherein the TGFβ superfamily ligand is selected from activin, GDF-11, and bone morphogenetic protein.

41. The composition according to claim 39 or 40 for the treatment of iron overload and normalization of red blood cell production in patients.

42. (i) A composition comprising a binder polypeptide according to any one of claims 1 to 28, and (ii) erythropoietin.

43. The composition according to claim 42 for the treatment of anemia associated with iron overload in a patient.

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