Treatment of Physiological Iron Excess

Abstract

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

Description

Technical Field

[0001] The present invention relates to an agent for reducing iron overload in patients having conditions such as beta-thalassemia and myelodysplastic syndrome (MDS).

Background Art

[0002] Iron is essential for erythropoiesis, i.e., the production of red blood cells that transport oxygen from the lungs to other tissues of the body. However, excess iron is toxic because of its ability to generate reactive oxygen species, and thus its absorption by the duodenum must be tightly regulated. The liver peptide hormone hepcidin has a central role in matching iron absorption to the body's iron requirements. Hepcidin negatively regulates cellular iron efflux by promoting the degradation of ferroportin, the only known iron exporter. Ferroportin is expressed in cells that are major storage compartments for iron, such as macrophages, and on the basolateral side of duodenal cells. Negative regulation of ferroportin by hepcidin thus limits duodenal iron absorption and the release of iron from iron-storage cells such as macrophages. It is well established that activation of the BMP-SMAD signaling pathway in hepatocytes via secretion of BMP ligands (mainly BMP6) stimulates hepcidin expression. Matriptase-2 (MTP-2), a type II transmembrane trypsin-like serine protease encoded by the gene Tmprss6, is expressed by hepatocytes and inhibits BMP-SMAD signaling by potentially cleaving BMP co-receptors such as HJV (Non-Patent Document 1), thereby reducing hepcidin expression and increasing dietary iron uptake and the release of iron from cellular stores. Expression of MTP-2 is induced by BMP6, which is essential for hepcidin expression, and by excess iron (Non-Patent Document 2). Thus, BMP6 stimulates hepcidin expression, but BMP6 also increases the expression of negative regulators that maintain a negative feedback mechanism to prevent abnormal regulation of iron (Non-Patent Document 3). Figure 1.

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

[0004] Beta-thalassemia is an inherited disorder of abnormal hemoglobin caused by genetic defects 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, and each globin subunit has a heme group with iron at its center that binds reversibly to oxygen. In beta-thalassemia, incomplete production of hemoglobin results in ineffective erythropoiesis and, as a result, anemia (a lack of oxygen-carrying red blood cells). Excessive production of erythropoietin (epo), which is upregulated in response to anemia, and / or an increase in the level of erythroferrone, an erythroid hormone resulting from abnormally increased and ineffective erythropoiesis, have an inhibitory effect on hepcidin, an iron regulatory factor, increasing iron absorption from the gastrointestinal tract and iron release from internal stores and causing iron overload. Interestingly, it is thought that iron overload, rather than anemia itself, shortens lifespan in beta-thalassemia. Increased iron availability increases transferrin saturation and heme production, which, along with a compensatory increase in alpha-globin expression, leads to the formation of hemichromes, as well as increased reactive oxygen species stress and apoptosis of erythroid progenitor cells. This highly stimulated but ineffective erythropoiesis results in the death of many mature red blood cells and, as a result, also causes splenomegaly or an enlarged 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, namely 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 transfusions ("transfusion-dependent beta thalassemia"). Patients with beta thalassemia intermedia, on the other hand, do not require regular 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] The unfortunate effects of transfusions in transfusion-dependent patients are that each transfusion contains at least 200 mg of iron and thus exacerbates the problems of iron overload and toxic tissue damage caused by transferrin-unbound iron, necessitating iron chelation therapy. Iron chelating agents can form complexes with transferrin-unbound iron in circulation, shift the equilibrium, and release iron from tissues to prevent tissue damage. However, chelating agents do not reduce transferrin saturation and therefore do not prevent the increased formation of hemichromes and increased apoptosis. Patients remain transfusion-dependent. Furthermore, iron chelating agents can have side effects such as renal failure, toxic neutropenia, and diarrhea. Transfusions and iron chelation have improved the prognosis of transfusion-dependent patients, but the iron overload that some patients had remains an unmet clinical need today.

[0007] Similarly, in the treatment of patients diagnosed with other iron overload anemias and related disorders, there is an unmet clinical need. Myelodysplastic syndromes (MDS) are a group of clonal stem cell disorders characterized by ineffective and dysplastic hematopoiesis that results in one or more cytopenias, as well as various tendencies to develop acute myeloid leukemia (AML). Some forms of MDS, including MDS with chromosome deletion 5q- (5q-MDS) and refractory anemia with ring sideroblasts (RARS), are associated with anemia and toxic iron deposition in erythroid precursors. These forms of MDS are often associated with reduced hepcidin levels. In many cases, MDS can be managed with regular transfusions, but these transfusions can lead to secondary iron overload and reduced overall survival, such as in beta thalassemia. MDS patients are therefore usually treated with iron chelating agents once a certain threshold of iron overload is reached.

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

[0009] Rare forms of anemia have also been shown in animal models to benefit from iron reduction therapy. These anemias include Blackfan-Diamond anemia and sickle cell anemia. Iron deposition in the liver (mainly parenchymal cells) has also been found to promote oxidative stress and fibrosis in diseases such as hemochromatosis and hepatitis C infection, as well as in iron-loaded anemia. Regulation of iron metabolism is also thought to be important in the development of liver fibrosis and cirrhosis, non-alcoholic fatty liver disease (NAFLD), and non-alcoholic steatohepatitis (NASH). Liver fibrosis often progresses to cirrhosis, which is associated with loss of liver function and progression to liver cancer.

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

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

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

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

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

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

[0016] Fusion proteins having the extracellular domain of the activin type II receptor bound to the Fc portion of human IgG1 are also in clinical trials. 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 luspatercept is an activin type IIB receptor IgG-Fc fusion protein. These proteins have been shown to significantly reduce the need for red blood cell transfusions in iron overload anemia, but to date, these proteins have not been proven to be sufficient to achieve transfusion independence (Non-Patent Document 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 more than a 50% reduction in transfusions over the same time frame. Therefore, there remains 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

Summary of the Invention

Problems to be Solved by the Invention

[0019] Perhaps the simplest concept of increasing hepcidin levels by therapeutic intervention was the therapeutic use of hepcidin, hepcidin derivatives, or analogs of these themselves (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 a simple treatment concept, none of these treatments have yet been able to meet the criteria for regulatory approval. At least two of these approaches have recently ended after clinical trials (LJPC - 401 in a Phase II trial in beta - thalassemia by La Jolla Pharmaceuticals, and M - 021 after a Phase I trial by Merganser). Here too, the medical need to develop effective new treatments still exists.

Means for Solving the Problems

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

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

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

[0023] As embodiments, antibodies referred to herein as NORI-001, NORI-002, NORI-003, NORI-004, NORI-005, NORI-006, NORI-007, NORI-008, NORI-009, NORI-010, NORI-011, NORI-012, NORI-013, NORI-014, NORI-015, NORI-016, NORI-017, NORI-018, NORI-019, NORI-020, NORI-021, NORI-022, NORI-023, NORI-024, NORI-025, NORI-026, NORI-027, NORI-028, NORI-029, NORI-030, NORI-031, NORI-032, and NORI-033 (collectively, "NORI-001 to NORI-033") are included. These correspond to various choices of the sequences of the antibody heavy and light chains. All of these antibodies have been demonstrated to bind to MTP-2 and inhibit its catalytic activity. Selected exemplary antibodies have also been shown to successfully reduce hepcidin expression in vivo, which is an important biological process in physiological iron overload and also indicates the potential ability of inhibitory anti-MTP-2 binder polypeptides to treat blood disorders and other conditions associated with iron overload.

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

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

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

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

[0028] In a first configuration, the antibody comprises a VH domain that includes the NORI-003 HCDR and has at least 90% sequence identity with the VH domain of NORI-003, and a VL domain that includes the NORI-003 LCDR and has at least 90% sequence identity with the VL domain of NORI-003 and includes.

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

[0030] In a second configuration, the antibody It includes a VH domain that contains NORI-006 HCDR and has at least 90% sequence identity with the VH domain of NORI-006, and a VL domain that contains NORI-006 LCDR and has at least 90% sequence identity with the VL domain of NORI-006 is included.

[0031] The antibody may include the NORI-006 VH domain and the NORI-006 VL domain. Optionally, the antibody is an IgG that includes the NORI-006 heavy chain and the NORI-006 light chain.

[0032] In a third configuration, the antibody includes a VH domain that contains NORI-008 HCDR and has at least 90% sequence identity with the VH domain of NORI-008, and a VL domain that contains NORI-008 LCDR and has at least 90% sequence identity with the VL domain of NORI-008 is included.

[0033] The antibody may include the NORI-008 VH domain and the NORI-008 VL domain. Optionally, the antibody is an IgG that includes the NORI-008 heavy chain and the NORI-008 light chain.

[0034] In a fourth configuration, the antibody includes a VH domain that contains NORI-011 HCDR and has at least 90% sequence identity with the VH domain of NORI-011, and a VL domain that contains NORI-011 LCDR and has at least 90% sequence identity with the VL domain of NORI-011 is included.

[0035] The antibody may include the NORI-011 VH domain and the NORI-011 VL domain. Optionally, the antibody is an IgG that includes the NORI-011 heavy chain and the NORI-011 light chain.

[0036] In various embodiments of the present invention, the % sequence identity of the VH and / or VL domain(s) can be, optionally, higher than 90%, for example, the % sequence identity can be 95% or higher, 98% or higher, or 99% or higher.

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

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

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

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

[0041] The effect of MTP-2 inhibition (e.g., reduction of hepcidin expression, measurable as reduction of the mRNA of its coding gene hamp) is further detected in vivo, and the inhibitory activity and biological relevance are confirmed.

[0042] In various embodiments, the potency of the inhibitor is quantified according to its IC50 measured in an in vitro assay for inhibition of MTP-2 enzyme activity (e.g., an assay as 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 comprising any of the VH and VL domains from 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 invention can have an IC50 within 25% or within 10% of the IC50 of any of NORI-001 to NORI-033, for example, NORI-003 IgG, NORI-006 IgG, NORI-008 IgG, NORI-009 IgG, or NORI-011 IgG, or a binder polypeptide according to the invention can have an IC50 lower than the IC50 of said reference antibody. "Within x% of" means that the IC50 of the test binder polypeptide is no greater than x% larger and no less than x% smaller than the IC50 of the reference antibody.

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

[0045] In one embodiment, the assay for MTP-2 inhibition is performed with human MTP-2. In another embodiment, the assay for MTP-2 inhibition is performed with non-human (e.g., mouse, rat, or cynomolgus monkey) MTP-2. In comparing the inhibitory efficacy of a binder polypeptide in the same enzyme assay using different species of MTP-2, an indication of the cross-species reactivity of the binder polypeptide is obtained. Generally, cross-species reactivity is desirable as it allows for testing of the binder polypeptide in vivo in multiple species. For example, preclinical studies in experimental animals (e.g., mouse, rat, or cynomolgus monkey) can be performed prior to clinical studies in the target species (e.g., human). Preferably, the binder polypeptide according to the present invention is cross-reactive with respect to binding and inhibition of MTP-2 of 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 that is within 50%, within 25%, or within 20% of its IC50 in an in vitro assay for inhibition of human MTP-2 enzyme activity in an in vitro assay for inhibition of non-human (e.g., mouse, rat, and / or cynomolgus monkey) MTP-2 enzyme activity.

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

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

[0049] The ability of the 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 an assay 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 region of MTP-2 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 with any anti-MTP-2 antibody described herein for binding to MTP-2. For example, an antibody comprising the VH and VL domains of any of NORI-001 to NORI-033 is used as the reference antibody. The reference antibody is provided as IgG. For example, in various embodiments, the binder polypeptide may compete with NORI-003 IgG, NORI-006 IgG, NORI-008 IgG, or NORI-011 IgG for binding to MTP-2. Alternatively, the reference antibody is provided as scFv. For example, in various embodiments, the binder polypeptide may compete with NORI-003 scFv, NORI-006 scFv, NORI-008 scFv, or NORI-011 IgG for binding to MTP-2.

[0050] IC50 can be determined in a competitive assay. For example, the binder polypeptide can 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] Nucleic acids encoding the binder polypeptides described herein are also provided as cells comprising said nucleic acids. In vitro host cells can contain the nucleic acid, which can optionally be integrated into the cellular (e.g., genomic) DNA of the host cell or transiently transfected (e.g., plasmid DNA).

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

[0053] Item Antibodies against the enzyme matriptase-2 (MTP-2) are presented. Inhibition of MTP-2 reduces dietary iron uptake and iron release from cellular stores in the body. Inhibitors of MTP-2 (such as antibodies against the serine protease domain) can be used to treat iron overload, which is characteristic of diseases such as beta-thalassemia and other diseases that cause toxic accumulation of iron. Combinations of MTP-2 inhibitors with activin receptor ligand traps or with erythropoietin provide additional therapeutic effects.

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

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

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

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

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

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

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

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

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

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

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

[0065] 11. In an enzymatic assay using the mouse MTP-2 extracellular domain, a binder polypeptide according to item 10, having an IC50 in said assay using the human MTP-2 extracellular domain that differs by less than 100-fold from its IC50.

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

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

[0068] 14. A binder polypeptide according to any one of items 1 to 13, which competes with an IgG containing any of the VH and VL domains from NORI-001 to NORI-033 for binding to human and / or mouse MTP-2.

[0069] 15. A binder polypeptide according to item 14, which competes with an 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 according to any one of items 1 to 15, which competes with aprotinin for binding to the serine protease catalytic domain of human and / or mouse MTP-2.

[0071] 17. A binder polypeptide according to 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. The binder polypeptide according to 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 according to 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. The binder polypeptide according to any one 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. The binder polypeptide according to any one of items 1 to 20, having an affinity (Kd) of less than 50 nM for mouse MTP-2 as determined by surface plasmon resonance.

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

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

[0078] 24. The VH domain and the VL domain are each a binder polypeptide according to item 23, obtained by recombination of a set of germline gene segments shown in Table G for 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.

[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 A 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 * 01 The binder polypeptide according to any one of items 1 to 25, comprising an antibody light chain variable (VL) domain obtained by recombination of 01.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0103] 47. Comprising the VH domain and the VL domain, which are the VH and VL domains of any one of NORI-001 to NORI-033, or comprising the VH and VL domains in which one or more non-germline residues of the framework region have reverted to the germline, An isolated antibody.

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

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

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

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

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

[0109] 53. The monoclonal IgG antibody comprising the heavy chain of NORI-003 antibody and the light chain of NORI-003 antibody, the heavy chain of NORI-006 antibody and the light chain of NORI-006 antibody, the heavy chain of NORI-011 antibody and the light chain of NORI-011 antibody, or the heavy chain of NORI-008 antibody and the light chain of NORI-008 antibody

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

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

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

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

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

[0115] 59. The composition according to any one of Items 56 to 58 for use in the treatment of the human or animal body by therapy.

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

[0117] 61. The combination according to Item 60 for use in the treatment of iron overload and normalization of erythropoiesis in a patient.

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

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

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

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

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

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

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

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

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

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

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

[0129] 72. In a patient, Reducing the absorption of dietary iron, Treating iron overload, Increasing the expression of hepcidin from hepatocytes, Reducing anemia caused by iron overload, Lowering the serum iron concentration and / or Lowering the iron saturation of transferrin A method comprising the step of administering to the patient a composition according to any one of items 56 to 58.

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

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

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

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

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

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

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

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

[0138] 81. The method according to item 72 or item 73, comprising the step of administering a further therapeutic agent, such as ruspatelcept, to the patient to reduce iron overload.

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

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

[0141] 84. In a patient Reducing the absorption of dietary iron, Treating iron overload, Increasing the expression of hepcidin from hepatocytes, Reducing anemia caused by iron overload, Lowering the serum iron concentration, and / or Lowering the iron saturation of transferrin A therapeutic agent for reducing iron overload for use in a method, the method comprising the step of administering the therapeutic agent and a composition according to any one of items 56 - 58 to a patient.

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

[0143] A therapeutic agent comprising an antagonist of a TGFβ family ligand for use in a method of promoting erythrocyte maturation in a patient, the method comprising the step of administering to the patient the therapeutic agent and the composition according to any one of items 56 to 58.

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

[0145] Matriptase-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 of MTP-2, TMPRSS6, is located at 22q12.3.

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

[0147] Similar to 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 additional endogenous proteins play important roles. MTP-2 is synthesized within the endoplasmic reticulum membrane and transported to the cell surface as an inactive enzyme precursor, by which MTP-2 autocleaves at an arginine residue within a highly conserved activation motif between the CUB2 domain and the serine protease. The MTP-2 serine protease domain has four essential disulfide bonds, one of which importantly attaches the domain to the backbone that is membrane-bound.

[0148] The cleaved form of MTP-2 exhibits an active form and mostly remains membrane-bound, in which case MTP-2 can cleave other membrane-bound targets on the cell surface. However, in the overexpression of MTP-2 in vitro, an "efflux-type" active form of MTP-2 has been found in the supernatant of cultured cells. Whether this efflux-type form is part of the native MTP-2 biology with in vivo functions or is merely the result of overexpression in a cell-based system remains unknown.

[0149] The amino acid sequence of MTP-2 is shown in Table S. For example, human MTP-2 has the amino acid sequence of Uniprot ID Q8IU80 and contains 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. An MTP-2 fragment containing the ECD, amino acids 78 - 811, can be produced recombinantly and is used in the assays described herein (e.g., in a histagged form). The generation of MTP-2 for use in the assays is detailed in Example 5.

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

[0151] MTP-2 referred to herein can 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] Since the processing of MTP-2 is complex and involves most of the protein structure, mutations in the protein can result in loss of function. In addition, TMPRSS6 polymorphisms such as rs855791 are known that can lead to increased MTP-2 activity and more efficient inhibition of hepcidin. The R576A mutation mutates an important arginine required for cleavage of the SP domain and full activation of MTP-2, thus maintaining the protein as an inactive enzyme precursor. The S762A mutation mutates an important serine residue within the catalytic triad of the SP domain, completely suppressing activity. Thus, since autoactivity is required for autocleavage, the protein also remains an inactive enzyme precursor here. The E114K mutant has been described in patients with expression of non-functional MTP-2. This mutation is present within the SEA domain and thus is highly likely to interfere with proper trafficking 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, which results in a single amino acid change from valine to alanine at position 736 (27.2%); A variant having both V736A and an additional K253E mutation (25.9%); and Variant with K253E alone (11.4%).

[0154] The binder polypeptide preferably binds to all four such variants and is thus suitable for treating all or most of the human population by inhibiting MTP-2 variants expressed by most of the human population. Thus, the binder polypeptide can bind to human MTP-2 containing sequence polymorphisms 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 testis. Isoform 2 is the major isoform in the liver and lacks the 9 N-terminal intracellular amino acids (802 amino acids). This N-terminal region is thought to be involved in the internalization of membrane-bound MTP-2 and thus internalizes more slowly than isoform 1. Isoform 3 is also mainly expressed in the testis together with isoform 1. This utilizes an alternative splicing variant of exon 10 that has the 9 N-terminal amino acids but lacks the SP domain and thus promotes the expression of a truncated form that is functionally inactive. Isoform 4 has the same exons as isoform 2 but also has an additional exon consisting of 22 amino acids between exons 16 and 17 that disrupts the function of the SP domain and is likewise functionally inactive. Isoform 4 is thought to be expressed in the tissues where isoform 2 is also expressed, and isoforms 3 and 4 are thought to be dominant negative regulators of isoforms 1 and 2 because their function is lost. It has been shown that the expression of isoform 3 or 4 can block the cleavage of HJV mediated by isoform 2.

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

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

[0158] The binder polypeptide can bind to the serine protease catalytic domain of MTP-2. The binder polypeptide can bind to auto-activated MTP-2. The binder polypeptide can bind to MTP-2 ECD. The binder polypeptide can bind to the MTP-2 enzyme precursor.

[0159] A binder polypeptide that binds to the serine protease catalytic domain is identified as one that binds to MTP-2 containing said domain and does not bind to MTP-2 lacking said domain. A "headless" variant of MTP-2 can be constructed that has a C-terminal truncation that deletes the serine protease domain but still contains the remaining portion of the ECD. Binding to the full-length MTP-2 ECD and the absence of binding to the headless MTP-2 ECD indicate 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 exemplary protocol is provided herein. Binding to the serine protease catalytic domain can also be identified in an enzyme inhibition assay using MTP-2 ECD and headless MTP-2 ECD. A binder that binds to the serine protease catalytic domain may show dose-dependent binding to MTP-2 ECD in such an assay and may not show dose-dependent binding to headless MTP-2 ECD in said assay.

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

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

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

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

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

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

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

[0167] Binding affinity The binder polypeptide may have an affinity (K D ) for human MTP-2 of less than 100 nM, less than 50 nM, less than 25 nM, or less than 10 nM. The binder polypeptide may have an affinity (Kd) for mouse MTP-2 of less than 100 nM, less than 50 nM, less than 25 nM, or less than 10 nM. The binder polypeptide may have an affinity (K D ) for rat MTP-2 of less than 100 nM, less than 50 nM, less than 25 nM, or less than 10 nM. The binder polypeptide may have an affinity (Kd) for 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 K of the binding to human MTP-2 Dcan 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. K D is optionally at least 0.001 nM, for example at least 0.005 nM.

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

[0170] The affinity of the binder polypeptide for 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) and 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). Example SPR procedures and conditions are shown in Example 6.

[0171] Briefly, SPR can be performed at 25°C by capturing the binder polypeptide on the chip for 60 seconds at a concentration of 1 μg / ml at 10 μl / min (approximately 35 - 50 RU is captured), and then injecting MTP-2 (analyte) for 120 seconds (association time) at 30 μl / min, and monitoring dissociation for 600 seconds. The analyte can 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 fit to a 1:1 interaction model (e.g., using Biacore evaluation software with globally fit Rmax, ka, kd, and RI = 0).

[0172] Quantification of affinity can be performed using SPR with a monovalent form of an antigen-binding polypeptide arm, such as an antibody Fab or Fv containing an antigen-binding site, or a heterodimeric immunoglobulin (e.g., IgG) having a single antigen-binding arm for the antigen of interest. Alternatively, it may be convenient to determine the affinity for a bivalent form of an antigen-binding polypeptide arm, such as IgG containing a homodimeric antigen-binding arm. SPR can involve (directly or indirectly) a coating dimer of the antigen-binding polypeptide arm on a biosensor chip, exposing the antigen-binding polypeptide arm to the antigen in buffer solutions of various concentrations, detecting the binding, and calculating the equilibrium dissociation constant KD of the binding interaction. SPR can be performed at 25 °C. A suitable buffer solution is 150 mM NaCl, 0.05% surfactant (e.g., P20), and 3 mM EDTA, pH 7.6. HBS-P 1× containing 2.5 mM CaCl2 (HEPES 10 mM, pH 7.4, NaCl 150 mM, EDTA 3 mM, 0.05% polysorbate 20, pH 7.6) is an example buffer. The binding 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 herein, the isolated and purified MTP-2 ECD is conveniently used in assays and is a suitable analyte for SPR.

[0174] Cross-reactivity Regulatory agencies may require demonstration of therapeutic efficacy of a candidate therapeutic molecule 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-react with the corresponding antigen derived from one or more non-human mammals. Thus, the binder can bind both non-human MTP-2 and human MTP-2.

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

[0176] An interspecies cross-reactive binding molecule can have a fold-difference in affinity for binding to human and non-human antigens of 100-fold or less, 50-fold or less, 30-fold or less, 25-fold or less, 20-fold or less, 15-fold or less, 10-fold or less, or 5-fold or less. In other words, the KD for binding of the extracellular domain of a human antigen can be within 30-fold, 25-fold, 20-fold, 15-fold, 10-fold, or 5-fold of the KD for binding of the extracellular domain of a non-human antigen.

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

[0178] The binding molecule can also be considered to have cross-species reactivity when the KD of the binding of both antigens meets a threshold, for example, when the KD of the binding to the human antigen and the KD of the binding to the non-human antigen are both 10 mM or less, preferably 5 mM or less, more preferably 1 mM or less. The KD can be 100 nM or less, 50 nM or less, 25 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, or 1 nM or less.

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

[0180] The binder polypeptide may have some ability to block enzyme activity via a fluorescence readout using MTP-2 substrates of multiple species (e.g., one or more or all of human and mouse, rat, and cynomolgus monkey MTP-2). The binder polypeptide can show dose-dependent inhibition of MTP-2 catalytic activity in the assays described herein using human and non-human (e.g., mouse, rat, or cynomolgus monkey) MTP-2.

[0181] Cross-species reactivity for the binding of antigens of different species can be advantageous, but selectivity of the binder for MTP-2 is still necessary to avoid unwanted side effects. Thus, in vivo, MTP-2 is preferably the only antigen to which the antigen-binding site of the binder polypeptide binds. Nevertheless, the binder polypeptide can optionally be engineered to contain additional binding sites, and antibodies containing antibody constant regions can optionally bind to, for example, one or more Fc receptors.

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

[0183] Inhibition of MTP-2 MTP-2 is mainly expressed in hepatocytes and plays a major role in iron metabolism by regulating the expression of hepcidin from hepatocytes. Currently, it has been revealed that the expression of hepcidin, a major regulator of iron homeostasis, is controlled by bone morphogenetic protein (BMP) growth factors, which bind to type I and type II BMP receptors present in hepatocytes and induce 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 and increases the secretion of hepcidin. Hepcidin binds to the iron transporter ferroportin present in duodenal cells, macrophages, and hepatocytes, induces internalization and degradation, and thus reduces the blood iron level by decreasing the amount of iron entering the blood. MTP-2 is thought to negatively regulate hepcidin expression by selectively cleaving members of the BMPR complex on the hepatocyte surface and thus silencing BMP / SMAD signaling. One proposed enzymatic target of MTP-2 is hemojuvelin (HJV), a coreceptor of the BMPR complex required for maximal BMP / SMAD signaling. However, although studies have shown that MTP-2 can cleave HJV and generate specific cleavage products, mice masked with MTP-2 showed a reciprocal decrease in the expression of membrane-bound HJV, while TMPRSS6 KO mice showed an increase in the level of cleaved HJV. More recently, studies have shown that MTP-2 is most likely to cleave multiple members of the BMPR complex to inhibit hepcidin expression.

[0184] According to a tissue-wide scan of TMPRSS6 mRNA expression, there is also low-level MTP-2 expression in the testis, but the role of MTP-2 here is mostly unknown. Due to the local expression of MTP-2, both TMPRSS6 KO mice and humans with loss-of-function MTP-2 mutations have greatly elevated iron levels and show a phenotype without further side effects, suggesting that its further role in iron regulation is limited and thus not a major problem for anti-MTP-2 therapy.

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

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

[0187] In various embodiments, the binder polypeptide can bind to the serine protease catalytic domain of MTP-2. The serine protease catalytic domain has the enzymatic active site of MTP-2. Inhibition can result from steric hindrance of the enzyme-substrate interaction by a binder polypeptide that binds to MTP-2 and partially or completely masks the enzymatic active site to reduce substrate binding. Inhibition can alternatively or additionally result from the binder polypeptide inducing inactivation of the conformational change in the serine protease catalytic domain or biasing the serine protease catalytic domain into an inactive conformation to reduce its enzymatic activity. Regardless of the molecular mechanism of inhibition, the ability of the binder polypeptide to inhibit MTP-2 serine protease activity can be functionally determined in an enzyme assay.

[0188] In this specification, in vitro assays for the inhibition of serine protease cleavage of MTP-2 substrates to produce detectable products are described. These include enzyme assays using purified MTP-2 ECD and fluorescent substrates, and enzyme assays using MTP-2 expressed on the cell surface and fluorescent substrates. 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, for example, at a final concentration of 50 μM. Binder polypeptides that are inhibitors of MTP-2 enzyme activity are identified by the dose-dependent inhibition of MTP-2 serine protease activity in such enzyme assays.

[0189] In one embodiment, the binder polypeptide has an IC50 of less than 100 nM in an enzyme assay against the human MTP-2 extracellular domain, having an activity rate of 0.075 U / μl in the presence of 50 μM Boc-Gln-Gly-Arg-AMC fluorescent substrate. The IC50 can be less than 80 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 25 nM, less than 20 nM, less than 15 nM, or less than 10 nM. The IC50 can optionally be at least 0.01 nM, at least 0.1 nM, at least 1 nM, at least 2 nM, at least 3 nM, or at least 5 nM.

[0190] In one embodiment, the binder polypeptide has an IC50 of less than 100 nM in an enzyme assay against a non-human (e.g., mouse, rat, or cynomolgus monkey) MTP-2 extracellular domain, having an activity rate of 0.075 U / μl in the presence of 50 μM Boc-Gln-Gly-Arg-AMC fluorescent substrate. The IC50 can be less than 80 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 25 nM, less than 20 nM, less than 15 nM, or less than 10 nM. The IC50 is, optionally, at least 0.01 nM, at least 0.1 nM, at least 1 nM, at least 2 nM, at least 3 nM, or at least 5 nM.

[0191] As described above, preferably, the binder polypeptide is interspecies cross-reactive and thus 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 an assay 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 fluorescent MTP-2 substrate at a final concentration of 50 μM. The binder polypeptide may have an IC50 of less than 100 nM in the assay. The IC50 may be less than 80 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 30 nM, less than 25 nM, less than 20 nM, less than 15 nM, or less than 10 nM. The IC50 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.

[0193] Inhibition of the enzymatic activity of MTP-2 is further detected in hepatocytes. For example, a binder polypeptide that inhibits MTP-2 may increase hepcidin expression in a hepatoma cell line (with or without bmp stimulation), which is measurable as an increase in hamp mRNA compared to a control.

[0194] Similar readings are obtained from in vivo assays. In mice to which the binder polypeptide is administered, inhibition of MTP-2 enzyme activity can result in an increase in hamp mRNA, a reduction in serum iron, and a reduction in transferrin saturation (TSAT). To measure these effects, the binder polypeptide is administered to wild type (e.g., at 10 mg / kg). The binder polypeptide can increase hamp mRNA in hepatocytes by at least 2-fold within 24 hours after administration, and this increase can persist 3 days, preferably 21 days, after administration (e.g., after a single 10 mg / kg dose administered intraperitoneally). The binder polypeptide can reduce the serum iron concentration in mice. Again, this can be detectable within 24 hours and preferably persists 3 days, preferably 21 days, after administration. Example experiments and protocols for measuring hamp mRNA and quantifying iron and TSAT in serum are shown in the Examples.

[0195] Binder polypeptide The binder polypeptide according to the present invention is a polypeptide molecule having the ability to bind to and inhibit MTP-2.

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

[0197] Preferably, the binder polypeptide of the present invention comprises an immunoglobulin domain in which the 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] The antibodies according to the present invention are immunoglobulins or molecules comprising immunoglobulin domains that are naturally or partially or wholly synthetically produced. The antibodies are 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, multispecific antibodies with a closed conformation, disulfide-bonded scfv, diabodies), which can be derived from any species that naturally produces antibodies or can be created by recombinant DNA technology; isolated from serum, B cells, hybridomas, transfectomas, yeast, or bacteria. The antibodies can be humanized using conventional techniques. The term antibody encompasses any polypeptide or protein that contains the antigen-binding site of an antibody. 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 being structural or functional. Functional epitopes are generally a subset of structural epitopes and have residues that are directly involved in the affinity of the interaction. Epitopes can also be conformational, i.e., can consist of non-linear amino acids. In certain embodiments, an epitope can include an antigenic determinant that is a chemically active surface group of a molecule such as an amino acid, sugar side chain, phosphoryl group, or sulfonyl group, and in certain embodiments, can have specific three-dimensional structural features and / or specific charge features.

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

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

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

[0203] A further example of an antibody is an H2 antibody that includes a dimer of heavy chains (5’-VH-(optional hinge)-CH2-CH3-3’) and lacks light chains.

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

[0205] Optionally, the binder polypeptide, or an antibody immunoglobulin domain thereof, can be fused or conjugated to an additional 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 antibodies of the invention can be multispecific antibodies, such as bispecific antibodies, that comprise (i) an antigen-binding site of the antibody to MTP-2 and (ii) an additional antigen-binding site that recognizes a different antigen (optionally, an antigen-binding site of an antibody described herein).

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

[0207] An antibody can comprise an antibody VH domain that includes CDR1, CDR2, and CDR3 of VH, as well as a framework. An antibody can alternatively or additionally comprise an antibody VL domain that includes CDR1, CDR2, and CDR3 of VL, as well as a framework. Examples of VH and VL domains and CDRs of antibodies according to the present invention are as listed in Table S. All VH and VL sequences, CDR sequences, CDR sets, as well as HCDR sets and LCDR sets disclosed herein are aspects and embodiments of the present invention. As described herein, a "CDR set" includes CDR1, CDR2, and CDR3. Thus, an HCDR set refers to HCDR1, HCDR2, and HCDR3, and an LCDR set refers to LCDR1, LCDR2, and LCDR3. Unless otherwise specified, a "CDR set" includes HCDR and LCDR.

[0208] As described in more detail in the Examples, the inventors isolated and characterized specific purpose antibodies called NORI-001, NORI-002, NORI-003, NORI-004, NORI-005, NORI-006, NORI-007, NORI-008, NORI-009, NORI-010, NORI-011, NORI-012, NORI-013, NORI-014, NORI-015, NORI-016, NORI-017, NORI-018, NORI-019, NORI-020, NORI-021, NORI-022, NORI-023, NORI-024, NORI-025, NORI-026, NORI-027, NORI-028, NORI-029, NORI-030, NORI-031, NORI-032, and NORI-033 (collectively "NORI-001 to NORI-033").

[0209] In various aspects of the present invention, unless otherwise specified in the context, an antibody can be selected from any of these antibodies or from a subset of NORI-003, NORI-006, NORI-011, or NORI-008.

[0210] The present invention encompasses anti-MTP-2 antibodies having the VH and / or VL domain sequences of all antibodies shown in the accompanying sequence listing and / or drawings, and antibodies comprising 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 another method such as Kabat. Unless otherwise specified, references to residues within the variable domain, or to CDRs or framework regions, refer to the IMGT definitions.

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

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

[0214] The present invention provides an antibody comprising any one of HCDR1, HCDR2, and / or HCDR3 of antibodies NORI-001 to NORI-033, and / or any one of LCDR1, LCDR2, and / or LCDR3 of these antibodies, for example a set of CDRs. The antibody may comprise a set of VH CDRs of one of these antibodies. Optionally, the antibody may also comprise a set of VL CDRs of one of these antibodies, and the VL CDRs may be derived from the same or a different antibody as the VH CDRs.

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

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

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

[0218] An antibody can include one or more CDRs, such as a set of CDRs, within the antibody framework. The framework region can be that of a human germline gene segment sequence. Thus, the antibody can be a human antibody having a VH domain that includes a set of HCDRs in a human germline framework. Typically, the antibody can also have a VL domain that includes a set of LCDRs, for example, in a human germline framework. An “antibody gene segment,” such as a VH gene segment, a D gene segment, or a JH gene segment, refers to an oligonucleotide having the nucleic acid sequence from which that portion of the antibody is derived. For example, a VH gene segment is an oligonucleotide that includes a nucleic acid sequence corresponding to a portion from FR1 to CDR3 of the polypeptide VH domain. Human V, D, and J gene segments are recombined to generate a VH domain, and human V and J segments are recombined to generate 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 joining domain or joining region of the antibody chain. Somatic hypermutation can generate an antibody VH or VL domain having a framework region that does not exactly match or align with the corresponding gene segment, but sequence alignment can be used to identify the closest gene segment and thus to identify from which specific combination of gene segments a particular VH or VL domain is derived. When aligning an antibody sequence with a gene segment, the amino acid sequence of the antibody is aligned with the amino acid sequence encoded by the gene segment, or the nucleotide sequence of the antibody is directly aligned with the nucleotide sequence of the gene segment.

[0219] The antibodies of the invention can be human antibodies or chimeric antibodies that include human variable regions and non-human (e.g., mouse) constant regions. The antibodies of the invention have, for example, human variable regions and optionally also have human constant regions.

[0220] Accordingly, the antibody may optionally include a constant region or a portion thereof, for example, a constant region or a portion thereof of a human antibody. For example, the VL domain may be attached at its C-terminus to an antibody light chain kappa or lambda constant domain. Similarly, the antibody VH domain may be attached at its C-terminus to all or part of an immunoglobulin heavy chain constant region (e.g., the CH1 domain or the 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 human heavy chain constant regions are shown in Table S.

[0222] Alternatively, the constant region of the antibody of the present invention can 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 fully human antibodies. Others are engineered to produce antibodies containing chimeric heavy chains and fully human light chains. If the antibody contains one or more non-human constant regions, such replacements may be made to produce an antibody more suitable for administration to humans as a therapeutic composition, since replacing them with human constant regions reduces their immunogenicity.

[0223] When an antibody is digested with the enzyme papain, two identical antigen-binding fragments, also known as "Fab" fragments, and an "Fc" fragment that has no antigen-binding activity but has the ability to crystallize are produced. As used herein, "Fab" refers to a fragment of an antibody that contains one constant domain and one variable domain of each of the heavy and light chains. The term "Fc region" as used herein is used to define the C-terminal region of an immunoglobulin heavy chain that includes the native sequence Fc region and variant Fc regions. The "Fc fragment" refers to the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of an antibody are determined by sequences in the Fc region, which is also the region recognized by Fc receptors (FcRs) found on certain types of cells. When an antibody is digested with the enzyme pepsin, an F(ab’)2 fragment is produced, which remains with the two arms of the antibody molecule bound together and contains two antigen-binding sites. The F(ab’)2 fragment has the ability to cross-link antigens.

[0224] "Fv," as used herein, refers to the smallest fragment of an antibody that retains both the antigen recognition site and the antigen-binding site. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain, tightly bound either non-covalently or covalently. 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. Collectively, 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 an antigen, although 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. As described in U.S. Patent No. 5,869,046 and U.S. Patent No. 6,121,022, which are incorporated herein by reference, the biological half-life can also be increased by modifying the CH1 domain or the CL region of the heavy chain constant region to have a salvage receptor binding epitope derived from two loops of the CH2 domain of the IgG Fc region. In another embodiment, the Fc hinge region of the antibody or antigen-binding fragment of the invention is mutated to decrease the biological half-life of the antibody or fragment. One or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment, such that the antibody or fragment has impaired Staphylococcus protein A (SpA) binding compared to native Fc-hinge domain SpA binding. Other methods of increasing serum half-life are known to those of skill in the art. Thus, in one embodiment, the antibody or fragment is pegylated. In another embodiment, the antibody or fragment is fused to an albumin binding domain, such as an albumin binding single domain antibody (dAb). In another embodiment, the antibody or fragment is PASylated (i.e., genetic fusion of a polypeptide sequence consisting of PAS (XL-Protein GmbH) that forms a non-charged random coil structure with a large hydrodynamic volume). In another embodiment, the antibody or fragment is XTENylated® / rPEGylated (i.e., genetic fusion of an imprecise repetitive peptide sequence (Amunix, Versartis) to a therapeutic peptide). In another embodiment, the antibody or fragment is ELPylated (i.e., genetic fusion to an ELP repeat sequence (PhaseBio)). These various half-life extending fusions are described in more detail in Strohl, BioDrugs (2015) 29:215-239, and for example, this fusion in Tables 2 and 6 is incorporated herein by reference.

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

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

[0228] An antibody may have a heavy chain constant region that binds to one or more types of Fc receptors but does not induce a cellular effector function, i.e., does not mediate ADCC, CDC, or ADCP activity. Such a constant region may be unable to bind to specific Fc receptors involved in causing ADCC, CDC, or ADCP activity.

[0229] The antibody can have a heavy chain constant region that does not bind to the Fcγ receptor. For example, the constant region can include an "E" mutation, such as the Leu235Glu mutation (i.e., where the wild-type leucine residue has mutated to a glutamic acid residue), also known as IgG4-E. Another optional mutation in the heavy chain constant region is Ser228Pro ("P" mutation), which increases stability by reducing Fab arm exchange. The heavy chain constant region can be an IgG4 that includes 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 can be an IgG4PE antibody that includes the sequence of the IgG4PE constant region shown in Table S.

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

[0232] An appropriate antibody constant region can be selected according to the genotype of the patient to be treated. For example, as described in US20160319017, a method for increasing erythropoiesis in a human patient is rs855791, rs2543519, rs2235324, and rs1421312 administering a binder (e.g., an antibody) that binds to human MTP-2 encoded by a TMPRSS6 nucleotide sequence comprising an SNP selected from the group consisting of; The binder is (a) the heavy chain constant region of human gamma-4 comprising Leu at position 189 or Arg at position 289 as shown in SEQ ID NO: 73 of US20160319017; and (b) the heavy chain constant region of human gamma-1 comprising Asp corresponding to position 204 of SEQ ID NO: 42 of US20160319017 or Leu corresponding to position 206 of SEQ ID NO: 42 of US20160319017 comprising a constant region selected from the group consisting of; wherein (i) the human subject comprises a TMPRSS6 nucleotide sequence comprising the selected SNP; and (ii) the human patient comprises a constant region gene segment encoding the selected constant region; or the human patient expresses an antibody comprising the selected constant region.

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

[0234] Additional example constant regions are shown in Table S.

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

[0236] For example, antibodies are generated by immunizing with MTP-2 or a fragment thereof (e.g., recombinant MTP-2 ECD) or its coding nucleic acid, and then optionally humanizing the constant region and / or variable region to produce human or humanized antibodies in experimental animals such as transgenic mice (e.g., KymouseTM, Velocimouse®, Omnimouse®, Xenomouse®, HuMab Mouse® or MeMo Mouse®), mice, rats (e.g., Omnirat®), camels, sharks, rabbits, chickens or other non-human animals. In one example, as will be apparent to those skilled in the art, display technologies such as yeast, phage or ribosome display are used. For example, standard affinity maturation using display technology is performed in a further step after isolating antibodies derived from transgenic animals, phage display libraries or other libraries. Representative examples of suitable techniques are described in US20120093818 (Amgen, Inc), which is incorporated herein by reference in its entirety, for example, the methods presented in paragraphs

[0309] to

[0346] .

[0237] There can be many reasons why it may be desirable to create variants of a binder, including optimization of the polypeptide sequence for large-scale manufacturing, facilitation of purification, enhancement of stability, or improvement of compatibility for encapsulation in a desired pharmaceutical formulation. For example, protein engineering efforts such as substituting one amino acid with an alternative amino acid (although Cys and Met may be exceptions and, optionally, generating variants containing all naturally occurring amino acids at this position) and monitoring the effects on function and expression to determine the best substitution are performed on one or more target residues of the antibody sequence. It may not be desirable to substitute a residue with Cys or Met, or to introduce these residues into the sequence, as doing so can cause problems in manufacturing, for example, by forming new intramolecular or intermolecular cysteine-cysteine bonds. When a primary candidate has been selected and optimized for manufacturing and clinical development, it will generally be desirable to leave its antigen-binding properties unchanged, or at least to retain the affinity and potency of the parental molecule. However, variants can also be generated to modulate important antibody characteristics such as affinity, cross-reactivity or neutralizing potency.

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

[0239] One or more amino acid mutations may optionally be introduced into the framework regions of the antibody VH or VL domains disclosed herein. For example, one or more residues different from the corresponding human germline segment sequences are reverted to the germline. The human germline gene segment sequences corresponding to the VH and VL domains of the exemplary anti-MTP-2 antibody are shown in Table G.

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

[0241] The alterations are made to one or more framework regions and / or one or more CDRs. Variants are optionally provided by CDR mutagenesis. The alterations generally do not result in a loss of function, and thus an antibody comprising an amino acid sequence altered in this way may retain the ability to bind to MTP-2. The antibody may retain the same quantitative binding ability as an antibody not subjected to the alterations, as measured, for example, in the assays described herein. An antibody comprising an amino acid sequence altered in this way may have an improved ability to bind to and / or inhibit MTP-2.

[0242] The modification may include replacing one or more amino acid residues with non-naturally occurring or non-standard amino acids, modifying one or more amino acid residues into non-naturally occurring or non-standard forms, or inserting one or more non-naturally occurring or non-standard amino acids into the sequence. Examples of the number and location of modifications in the sequences of the present invention are described elsewhere in the present invention. Naturally occurring amino acids include the 20 "standard" L-amino acids, identified by the standard one-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 can be incorporated into the polypeptide backbone or result from the modification of existing amino acid residues. Non-standard amino acids may or may not occur naturally.

[0243] As used herein, the term "variant" 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 changes 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 with respect to any of polarity, side chain functionality, or size. Such conservative substitutions are well known in the art. The substitutions encompassed by the present invention may also be "non-conservative" in which case the amino acid residue present in the peptide is replaced with an amino acid having different characteristics, e.g., a naturally occurring amino acid from a different group (e.g., replacing a charged or hydrophobic amino acid with alanine), or alternatively, a naturally occurring amino acid is replaced with a non-conventional amino acid. In some embodiments, the amino acid substitutions are conservative. Also, when used with respect to a polynucleotide or polypeptide, the polynucleotide or polypeptide encompassed within the term variant refers to a polynucleotide or polypeptide that may have a different primary, secondary, or tertiary structure as compared to a reference polynucleotide or polypeptide (e.g., as compared to a wild-type polynucleotide or polypeptide).

[0244] In some embodiments, “synthetic variants,” “recombinant variants,” or “chemically modified” polynucleotide or polypeptide variants isolated or generated using methods well known in the art can be used. “Modified variants” may include conservative or non-conservative amino acid changes as described below. Polynucleotide changes can result in amino acid substitutions, additions, deletions, fusions, and truncations in the polypeptide encoded by the reference sequence. Some embodiments use insertion variants, deletion variants, or substitution variants by amino acid substitution, including insertions of amino acids and other molecules not normally occurring in the underlying peptide sequence of the variant, such as, but not limited to, the insertion of ornithine, which does not normally occur in human proteins. The term “conservative substitution,” when referring to a polypeptide, refers to a change in the amino acid composition of the polypeptide that does not substantially change the activity of the polypeptide. For example, a conservative substitution refers to substituting an amino acid residue with 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 the exchange of leucine for isoleucine or valine, aspartic acid for glutamic acid, or threonine for serine. Tables of conservative substitutions that provide functionally similar amino acids are well known in the art. For example, the following six groups each contain amino acids that are conservative substitutions 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 typically range from about 1 to 5 amino acids. The selection 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 as compared to an amino acid that is localized internally and not exposed to the solvent), an amino acid to substitute the existing amino acid can be selected. The selection of such conservative amino acid substitutions is well known in the art as disclosed, for example, in Dordo et al., J. Mol Biol, 1999, 217, pp. 721 - 739, and Taylor et al., J. Theor. Biol. 119 (1986); 205 - 218, and S. French and B. Robson, J. Mol. Evol. 19 (1983) 171. Thus, conservative amino acid substitutions suitable for amino acids outside the protein or peptide (i.e., 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 amino acids inside the protein or peptide, for example, conservative substitutions that are appropriate for amino acids inside the protein or peptide (i.e., amino acids that are not exposed to the solvent) can be used. For example, but not limited to, the following conservative substitutions can be used: Y is replaced by F, T is replaced by A or S, I is replaced by L or V, W is replaced by Y, M is replaced by L, N is replaced by D, G is replaced by A, T is replaced by A or S, D is replaced by N, I is replaced by L or V, F is replaced by Y or L, S is replaced by A or T, and A is replaced by S, G, T or V. In some embodiments, non-conservative amino acid substitutions are also included within the term variants.

[0247] The present invention includes a method of making an antibody containing a VH and / or VL domain variant of the antibody VH and / or VL domains shown in Table S. Such an antibody (i) wherein the parental antibody VH domain is a VH domain comprising any of the VH domains from NORI-001 to NORI-033, or a VH domain comprising any of the heavy chain complementarity determining regions of those antibodies, providing an antibody VH domain that is an amino acid sequence variant of the parental antibody VH domain by addition, deletion, substitution or insertion of one or more amino acids into the amino acid sequence of the parental antibody VH domain; (ii) optionally combining the VH domain thus provided with a VL domain to provide a VH / VL combination; (iii) testing the VH domain or VH / VL domain combination thus provided to identify an antibody having one or more desired characteristics can be made by a method comprising.

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

[0249] Desired characteristics include binding to human and / or non-human MTP-2. Antibodies are identified that have equal or higher affinity for human and / or mouse MTP-2 compared to the parental antibody. Other desired characteristics include inhibition in the enzyme assays described herein, as well as in vivo reduction of serum iron concentration and / or TSAT, and increase in hamp mRNA. Identification of antibodies having the desired characteristics can include identification of antibodies having functional attributes described herein, such as their affinity, cross-reactivity, specificity, or neutralizing titer, any of which can be determined by the assays described herein.

[0250] If the VL domain is included in the method, the VL domain may be any of the VL domains from NORI-001 to NORI-033, or may be a variant provided by addition, deletion, substitution, or insertion of one or more amino acids to the amino acid sequence of the parental VL domain, where the parental VL domain is any of the VL domains from NORI-001 to NORI-033, or a VL domain that includes any of the light chain complementarity determining regions 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] Methods for generating variant antibodies may optionally include the step of making copies of the antibody or VH / VL domain combinations. The method may further include the step of expressing the resulting antibody. Nucleotide sequences corresponding to the desired antibody VH and / or VL domains can optionally be made in one or more expression vectors. Suitable methods of expression, including recombinant expression in host cells, are described in detail herein.

[0252] Encoded Nucleic Acid and Production Method Isolated nucleic acids encoding the antibodies according to the invention are provided. The nucleic acids may be DNA and / or RNA. Genomic DNA, cDNA, mRNA or other RNA of synthetic origin, or any combination thereof, can encode the antibody.

[0253] The invention also 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 the nucleotide sequences described herein include, unless the context specifically requires otherwise, DNA molecules having the specified sequences and RNA molecules having the specified sequences in which U is substituted for T.

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

[0255] Systems for the cloning and expression of polypeptides in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, plant cells, filamentous fungi, yeast and baculovirus systems, 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 fetal kidney cells (e.g., HEK293), human fetal retinal cells and many other cells.

[0257] The vector may contain appropriate control sequences, including, if necessary, a promoter sequence, a terminator sequence, a polyadenylation sequence, an enhancer sequence, a marker gene, and other sequences. A nucleic acid encoding an antibody is introduced into a host cell. The nucleic acids of the present invention can be integrated into the genome (e.g., chromosome) of the host cell. Integration can be facilitated by including sequences that promote recombination with the genome according to standard techniques. Nucleic acids can be introduced into eukaryotic cells by a variety of methods including calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses such as vaccinia or, for insect cells, baculovirus. Introduction of nucleic acids into host cells, particularly eukaryotic cells, may use a virus or plasmid-based system. Plasmid systems are maintained as episomes or integrated into the host cell or artificial chromosome. Integration may be by either random integration or targeted integration of one or more copies at single or multiple loci. For bacterial cells, suitable techniques include calcium chloride transformation, electroporation, and transfection using bacteriophage. Following introduction, the host cells are cultured, for example, under conditions for expression of the gene to express the nucleic acid, and then optionally, a binder polypeptide, such as an antibody, is isolated or purified.

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

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

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

[0261] The present invention provides a therapeutic composition comprising a binder polypeptide described herein. Also provided are therapeutic compositions comprising a nucleic acid encoding such a binder polypeptide. The encoding nucleic acids are described in more detail elsewhere in the present invention and include DNA and RNA, such as mRNA. In the treatment methods described herein, the use of a nucleic acid encoding a binder polypeptide and / or the use of cells containing such a nucleic acid can be used in place of (or in addition to) a composition comprising the binder polypeptide itself. Cells containing a nucleic acid encoding a binder polypeptide, optionally with the nucleic acid stably integrated into the genome, thus constitute a pharmaceutical for therapeutic use in a patient. The nucleic acid encoding the binder polypeptide is introduced into human cells derived from the intended patient and modified ex vivo. Administration of the cells containing the encoding nucleic acid to the patient provides a reservoir of cells capable of expressing the binder polypeptide and can provide a longer-term therapeutic benefit compared to administration of an isolated nucleic acid or an isolated binder polypeptide. The nucleic acid can also be administered directly to the patient for gene therapy. Thus, a nucleic acid encoding a binder polypeptide is provided for use in gene therapy, which involves introducing the encoding nucleic acid into the patient's cells in vivo such that the nucleic acid is expressed in the patient's cells to provide a therapeutic effect. Examples of therapeutic effects include an increase in hamp mRNA, a decrease in serum iron, a decrease in TSAT, and the treatment of diseases and conditions associated with iron overload, as 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 (trademark)), 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 pharmaceutical injection buffer and / or an adjuvant.

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

[0264] A variety of delivery systems are known and can be used to administer the pharmaceutical composition of the invention. Routes 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 (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active agents. Administration can be systemic or local. The antigen - binding molecule is preferably administered by subcutaneous injection. Administration can be self - administration by the patient, for example, self - injection.

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

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

[0267] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injections, intravenous drip, etc. These injectable preparations can be manufactured by known methods. For example, an injectable preparation can be manufactured by dissolving, suspending, or emulsifying the antibody or its salt described above, for example, in a sterile aqueous medium or an oily medium customarily used for injection. Examples of aqueous media for injection include physiological saline, isotonic solutions containing glucose, and other adjuvants, which are used in combination with appropriate solubilizing agents such as alcohols (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. Examples of oily media include sesame oil, soybean oil, etc., which are used in combination with solubilizing agents such as benzyl benzoate, benzyl alcohol, etc. The injection thus manufactured can be filled into appropriate ampoules. The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using standard needles and syringes. The treatment is not assumed to be limited to use in a clinic. Therefore, subcutaneous injection using a needleless device is also advantageous. Regarding subcutaneous delivery, pen-type delivery devices can be easily applied to the delivery of the pharmaceutical composition of the present invention. Such pen-type delivery devices can be reusable or disposable. Reusable pen-type delivery devices generally utilize replaceable cartridges containing the pharmaceutical composition. When 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. In a disposable pen-type delivery device, there is no replaceable cartridge. Instead, a disposable pen-type delivery device is pre-filled with the pharmaceutical composition held in a reservoir within the device. When the pharmaceutical composition in the reservoir is empty, 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, but are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Burgdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, Ind.), NOVOPEN™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, N.J.), OPTIPENT™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIKT™ (Sanofi-Aventis, Frankfurt, Germany). Examples of disposable pen-type delivery devices suitable for subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, the SOLOSTAR™ pen (Sanofi-Aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly).

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

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

[0270] One aspect of the invention is a composition comprising a binder polypeptide or nucleic acid of the invention and one or more pharmaceutically acceptable excipients, examples of which are listed above. "Pharmaceutically acceptable" means approved or approvable by a regulatory agency of the federal or state government of the United States with respect to use in animals, including humans, or listed in the United States Pharmacopeia or other generally recognized pharmacopeias. A pharmaceutically acceptable carrier, excipient, or adjuvant can be administered to a patient together with a binder polypeptide, for example, any antibody or polypeptide molecule described herein, without impairing its pharmacological activity and is non-toxic when administered in a dosage sufficient to deliver a therapeutically effective amount of the agent.

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

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

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

[0274] Administration is usually at a "therapeutically effective amount", which is an amount that produces the desired effect sufficient to demonstrate a benefit to the patient and for which it is administered. The exact amount will depend on the purpose of the treatment and can be ascertained by one of ordinary skill in the art using known methods (see, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding). The determination of the treatment prescription, such as the dosage, etc., is within the scope of the responsibility of the general practitioner and other physicians and may depend on the symptoms of the disease being treated and / or the severity of the progression. The therapeutically effective amount or appropriate dosage of the binder polypeptide or nucleic acid can be determined by comparing its in vitro and in vivo activities in animal models. Methods for extrapolating effective dosage amounts for humans in mice and other test animals are known.

[0275] In the treatment methods described herein, one or more dosages are administered. In some cases, a single administration may be effective in achieving long-term benefits. Thus, the method may include a single administration of the binder polypeptide, its encoding nucleic acid, or composition. Alternatively, multiple administrations are usually given continuously and at intervals of several days, weeks, or months. For example, the administrations may be every two weeks, every three weeks, or every four weeks. Optionally, the binder polypeptide is 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 a therapeutic treatment that is intended to reverse, alleviate, improve, inhibit, slow down, or stop the progression or severity of a condition associated with a disease or disorder. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease, or disorder. A treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, a treatment is "effective" if the progression of a disease is reduced or stopped. That is, "treatment" includes not only an improvement in symptoms or markers, but also halting, or at least slowing, the progression or worsening of symptoms as compared to what is expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms (whether detectable or undetectable), diminution of the extent of a disease, stabilization (i.e., not worsening) of the state of a disease, delay or slowing of disease progression, improvement or palliation of the state of a disease, remission (whether partial or complete), and / or decrease in mortality. The term "treatment" of a disease also includes providing relief from the symptoms or side effects of the disease (including palliative treatment). For effective treatment, complete cure is not contemplated. The methods may also include cure in certain embodiments. In the context of the present invention, a treatment may be a prophylactic treatment.

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

[0278] A binder polypeptide is provided that is administered at regular intervals of one week, two weeks, three weeks, four weeks, or one month.

[0279] Therapeutic use Therapeutic applications addressed by the present invention include the treatment of patients in which it is beneficial to inhibit MTP-2 activity and / or reduce iron uptake. Particular therapeutic areas are anemias characterized by high iron loading and deposition and inadequate erythropoiesis. As discussed previously, some forms of anemia often occur in association with primary iron overload (caused by inappropriately low hepcidin levels due to high erythropoietic activity) and ineffective erythropoiesis characterized by secondary iron overload mainly due to repeated red blood cell transfusions. Iron overload contributes to anemia by adversely affecting organ function, negatively affecting erythropoiesis, increasing heme and ROS in erythroblast progenitor cells and driving apoptosis, resulting in a near absence of functional red blood cells. Iron reduction reduces apoptosis, improves alpha / beta globin imbalance, and enables the production of more mature red blood cells. Reduced iron utilization capacity in erythroid cells results in reduced heme production, increased maturation of erythroid precursors, and increased hemoglobin levels. Thus, in some conditions, the therapeutic mechanism of action of the binder polypeptide may be inhibition of the enzymatic activity of MTP-2, which increases the level of hepcidin, resulting in iron restriction that normalizes erythropoiesis and improves the quality of hemoglobin and red blood cells.

[0280] Treatment of anemia, as well as addressing the toxic effects of iron overload, can similarly improve cardiac function and reduce fatigue.

[0281] The effects of the treatment according to the present invention include the following: Reduction of iron absorption by diet, Treatment of iron overload, Increase in the expression of hepcidin from hepatocytes, Reduction of anemia due to iron overload, Decrease in serum iron concentration, Decrease in transferrin iron saturation, Reduction in the need for blood transfusion, Reduction in the need for iron chelation therapy, Prolongation of survival, and / or Normalization of erythropoiesis.

[0282] The treatment may be beneficial in many conditions including the following discussed herein: Myelodysplastic syndromes (MDS) with a lower risk of having ring sideroblasts (RARS) that require blood transfusion 5q-MDS; Transfusion-dependent beta-thalassemia or severe beta-thalassemia; Transfusion-independent beta-thalassemia or intermediate beta-thalassemia; For example, hemochromatosis in patients without mutations in HJV or hepcidin, or hemochromatosis type 1 or 3; Cirrhosis; Fatty liver; For example, liver 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-related anemia (CKD).

[0283] Thus, a patient treated according to the present invention may have one of the above conditions. The treatment method may include administering to the patient a binder polypeptide, nucleic acid or composition described herein. Examples of formulations and administration methods are described elsewhere in the present invention. Depending on the disease state, the treatment 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] Increased hepcidin levels by treatment with an anti-MTP-2 binder polypeptide are thought to lead to a reduction in heme and hemichrome production in beta-thalassemia, or a reduction in TSAT levels accompanied by a reduction in the appearance of ring sideroblasts in MDS. This will result in a decrease in apoptosis of erythroid precursors and a higher blood cell count. Although the hemoglobin content of a single cell will decrease, the total hemoglobin level will be higher due to the increase in the number of red blood cells. This will then reduce the need for red blood cell transfusions.

[0285] Avoidance of, or reduction in the risk of, toxic tissue iron overload should increase the overall survival of the patient, i.e., extend the survival of the treated patient. Avoiding or reducing the transfusion burden, and / or avoiding or reducing the need for iron chelation and / or phlebotomy should also improve the quality of life of the patient.

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

[0287] Interestingly, the Tmprss6 gene polymorphism 736 V(A)->A(G) has been reported to be associated with higher hepcidin levels, reducing iron overload and improving 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 Tmprss6 and / or increased hepcidin in a mouse liver fibrosis model.

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

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

[0290] Treatment with the binder polypeptide according to the present invention can be combined with one or more additional treatments, for example, additional therapeutic agents for treating iron overload. The binder polypeptide can be combined with an activin type II receptor agonist fusion protein, for example, luspatercept. Since the MTP-2 inhibitor provides a mechanism of action that is alternative to other planned and existing treatments, it may provide a synergistic effect on erythropoiesis. A combination of (i) an inhibitor of MTP-2 and (ii) an antagonist of a TGFβ superfamily ligand, for example, a receptor-based ligand scavenger / trap of the TGFβ superfamily, can therefore result in an advantageous therapeutic effect.

[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 created by providing the extracellular domain of the receptor in a soluble form, where the receptor retains its ability to bind its ligand in the extracellular domain but does not induce downstream signaling (which would otherwise result from normal receptor:ligand interactions). The extracellular domain of the receptor can be linked to an Fc region to form a fusion protein.

[0292] Preferably, the receptor is an activin II receptor, such as ActIIRA or ActIIRB. The antagonist may be a polypeptide comprising a soluble extracellular domain of an activin II receptor, such as activin receptor IIB (ActRIIB), fused to an Fc region. Luspatercept 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 Acceleron Pharma's US7988973, which is incorporated herein by reference. Suragani RN et al. previously described a modified human ActRIIB extracellular domain (residues 24-131 of the native precursor cell with an L79D substitution) linked to the mouse IgG2a Fc domain, "RAP-536", and it was reported to reduce ineffective erythropoiesis and disease complications in mouse beta-thalassemia (Blood 123(25):3864-3872, 2014). Sotatercept is an example of an activin type A receptor IgG-Fc fusion protein. Antagonists of other TGFβ family ligands, such as BMPR-Fc fusion proteins, can be generated similarly.

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

[0294] Antagonists of TGFβ family ligands such as ActRIIB-Fc, ActIIRIIA-Fc or BMPR-Fc promote the maturation of erythroid progenitor cells during erythropoiesis, while MTP-2 inhibitors result in a decrease in iron (helping to normalize pathologically high iron levels) and thus delay erythropoiesis. From the tests reported herein (see Example 22), the inventors believe that the combination of these two actions results in a more efficient production of more mature erythrocytes. The inventors have demonstrated, for example, that the combination of an MTP-2 inhibitor (represented by NORI-11-M) and an activin receptor II ligand trap (represented by ActRIIB-Fc) is more effective than the ligand trap alone and corrects both iron overload and related anemia. The combination with an MTP-2 inhibitor can expand the therapeutic ability of TGFβ superfamily ligand traps by providing a greater therapeutic effect in patients (e.g., non-transfusion-dependent β-thalassemia patients) who already have a condition for which TGFβ superfamily ligand traps are already indicated. The combination with an MTP-2 inhibitor can also expand the therapeutic ability of ligand traps to treat additional patient populations such as patients with severe β-thalassemia, where treatments such as luspatercept currently have limited benefit.

[0295] Luspatercept has recently been approved by the FDA for myelodysplastic syndromes. Ineffective erythropoiesis suppresses hepcidin production in the liver, thus leading to unrestricted intestinal iron uptake, and iron overload begins to occur in MDS patients before they become transfusion-dependent. Transfusions then exacerbate the iron overload. The same situation occurs in transfusion-dependent β-thalassemia patients. In such patients treated with luspatercept (or other TGFβ superfamily ligand traps), the therapeutic benefit will be improved by including an MTP-2 inhibitor in the treatment regimen. The combination therapy can be used for any of the therapeutic applications or conditions described herein, for example, Diamond-Blackfan anemia.

[0296] The inhibitor of MTP-2 for use in combination therapy may be the binder polypeptide described herein, or another type of molecule such as a nucleic acid inhibitor of TMPRSS6 expression (e.g., an antisense or siRNA molecule targeting TMPRSS6) or a small molecule inhibitor (e.g., 3-amidinophenylalanine-derived matriptase-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, Matis G, Zs N, Steinmetzer T, Paszti-Gere E (2016) The impact of acute matriptase inhibition in hepatic inflammatory models. Biomed Res Int. https: / / doi.org / 10.1155 / 2016 / 6306984). Thus, the patient to be treated according to the present invention may also be a patient who receives treatment with an additional therapeutic agent for reducing iron overload. The method may include co-administering the binder polypeptide and the additional therapeutic agent to the patient, optionally in separate formulations. The compositions are administered sequentially or simultaneously. The same applies when a small molecule or nucleic acid MTP-2 inhibitor is used instead of the binder polypeptide. That is, the patient is treated either simultaneously or sequentially with a combination of an MTP-2 inhibitor and an additional therapeutic agent. The MTP-2 inhibitor and the additional therapeutic agent are preferably provided in separate formulations and administered separately. Generally, sequential administration is carried out on the same day (optionally separated by a period of minutes or hours) or on different days.

[0297] Treatment with an MTP-2 inhibitor (e.g., a binder polypeptide described herein) can be combined with erythropoietin (epo). Darbepoetin alfa, commercially known as ARANESP, is a structurally re-engineered form of epo that has an extended drug half-life compared to standard epo alpha and epo beta proteins. Darbepoetin alfa is used to stimulate erythropoiesis in anemic patients to increase hemoglobin levels and reduce the need for blood transfusions. The drug is administered at different doses depending on the severity of anemia, but in patients with anemia associated with chronic kidney disease (CKD), the recommended starting dose is 0.45 mcg / kg iv / sc every 4 weeks until the hemoglobin level reaches above 10 g / dL, and the dose is then reduced when the level reaches above 10 g / dL. ARANESP has been shown to improve hemoglobin levels in a small-scale trial in patients with intermediate beta-thalassemia (Singer et al 2011), but generally ARANESP is not a treatment option for beta-thalassemia patients due to unacceptable increases in RBC apoptosis and accompanying splenomegaly. As the results presented herein show, the therapeutic benefit 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 performed simultaneously every 2 - 4 weeks. Based on the results obtained in a beta-thalassemia mouse model (see Examples 20 and 21), such a co-treatment strategy should result in a therapeutic improvement in anemia from ARANESP treatment while maintaining spleen size and reducing concomitant toxic iron overload brought about by anti-MTP2 treatment. The exact co-administration regimen will be investigated and optimized for humans, but generally will involve administration of the binder polypeptide according to the present invention to the patient, and similarly administration of erythropoietin (preferably recombinant erythropoietin or a medically approved variant thereof, e.g., darbepoetin alfa). Optionally, the binder polypeptide and epo are administered simultaneously. Alternatively they are administered sequentially (on the same day or on separate days).Optionally, the binder polypeptide and epo are administered subcutaneously by separate or mixed injections. In some embodiments, the invention provides for the treatment of a patient with epo and a binder polypeptide, for example, in a patient having a condition described herein such as beta-thalassemia, administration of the binder polypeptide reduces erythrocyte apoptosis and the accompanying splenomegaly associated with the administration of epo. The binder polypeptide can thus be used to normalize erythropoiesis in patients being treated with epo.

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

Brief Description of the Drawings

[0299]

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Figure 18-2

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Example

[0300] Antibodies targeting MTP-2 for the treatment of iron overload diseases are described herein. By immunizing transgenic mice that generate antibodies with human variable domains and testing a wide variety of antibodies in a series of biologically relevant assays, species-cross-reactive MTP-2-specific monoclonal antibodies (mAbs) that are cross-reactive neutralizers of MTP-2 enzyme activity both in vitro and in vivo were obtained. The inventors have shown that the selected mAbs increase the level of hepcidin expression from liver cells after a single dose. The increase in hepcidin decreases serum iron and transferrin saturation by increasing the internalization and degradation of ferroportin. The Hbb model of beta-thalassemia th3 / +In mice, a single dose of 10 mg / kg resulted in a 52% and a 47% decrease in serum iron and transferrin saturation, respectively, at the 2-week time point. Further, by repeating the dosing, the inventors observed consistent iron restriction over multiple weeks in mice. These results indicate the potential ability of such mAbs to treat patients with iron overload, reduce the patients' anemia, and decrease the need for transfusion and iron chelation. th3 / + In mice, consistent iron restriction over multiple weeks was observed. These results indicate the potential ability of such mAbs to treat patients with iron overload, reduce the patients' anemia, and decrease the need for transfusion and iron chelation.

Example 1

[0301] Generation of a panel of anti-MTP-2 inhibitory antibodies Kymab transgenic mice that produce antibodies with human variable domains were immunized with MTP-2 using various different immunization regimens and antigen constructs, and antigen-specific B cells were selected. See Lee et al., Nat Biotechnol 32(4):356 - 63 2014; WO2011 / 004192; WO2011 / 158009, and WO2013 / 061098. The antibodies were tested in protein-based and cell-based in vitro assays for binding to human and mouse MTP-2, as well as for the 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 the recovered antibodies to the purified MTP-2 extracellular domain (ECD), and subsequently to confirm the binding to MTP-2 expressed on the cell surface.

[0303] Cross-reactive antibodies were selected in terms of the 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 human and mouse purified MTP-2 ECD in solution, as evaluated in an enzymatic assay containing a chromogenic MTP-2 substrate, and their ability to inhibit the enzymatic activity of human MTP-2 expressed in HEK293 cells.

[0305] A number of diverse antibodies were obtained, including examples of antibodies that bound to and inhibited human MTP-2 but did not bind to or inhibit mouse MTP-2, and other antibodies that bound to and inhibited mouse MTP-2 but did not bind to or inhibit human MTP-2. From the perspective of potential development as a cross-reactive inhibitor of MTP-2 activity, the antibodies described in Table G below were selected as particularly interesting antibodies.

[0306] [Table 1] Example 2

[0307] Antibody sequences The sequences of the HCDR, LCDR, VH domain, and VL domain of each of antibodies NORI-001 to NORI-033 are shown in Table S. The complete IgG4PE heavy chain of each antibody is shown. The complete light chain of each antibody is also shown. Unless otherwise indicated in 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 be indicated; for example, "NORI-001 IgG" is an IgG having the NORI-001 VH domain and the NORI-001 VL domain, and "NORI-001 scFv" is an scFv having the NORI-001 VH domain and the NORI-001 VL domain.

[0308] Antibody NORI-002 was confirmed to have a high-risk free cysteine liability in the VH domain. This amino acid was thought to be buried by analysis with structural software, but was mutated to mitigate any potential liability. Therefore, the C49C mutation was introduced into NORI-002, and the new antibody containing this mutation was designated NORI-003.

[0309] Antibody NORI-010 is a sequence optimized by introducing the P124S mutation into the VH domain for improved stability and expression, thereby generating a new antibody, NORI-011.

Example 3

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

[0311] All of antibodies NORI-001 to NORI-034 inhibited the enzyme activities of both human and mouse MTP-2 in this assay. The IC50 values for the inhibition of human MTP-2 were in the range of approximately 1.5 - 55 nM. The IC50 values for the inhibition of mouse MTP-2 were in the range of approximately 0.48 - 40 nM. Table D. Figure 3.

[0312]

Table 2-1

Table 2-2

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

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

[0315] 20 μl of titrated control and diluted antibody were plated in 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, due to the difference 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 enzyme reaction was allowed to proceed at room temperature, and then the fluorescence activity was read at 360 nm excitation wavelength and 460 nm emission wavelength in a plate reader (Envision) at 30 minutes, 1 hour, and 2 hours. When the substrate peptide is cleaved via the enzyme activity of MTP-2, the 7-amido-4-methylcoumarin (AMC, MCA, or NHMec) moiety is released from the C-terminus. AMC is a fluorophore incorporated into carboxypeptidase substrates at the C-terminus. Subsequently, excitation of the released coumarin occurs by excitation at 360 - 380 nm using a spectrofluorometer, and emission at 440 - 460 nm can be detected. 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 entered into graphpad software to create a logarithmic curve, and IC50 values were generated using non-linear regression parameters and the logarithmic (inhibition) vs. response - variable slope (four-parameter) equation.

[0316] For details on the preparation of the antigen reagent, see Example 5.

Example 4

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

[0318] Antibodies NORI-008 to NORI-010, NORI-012 to NORI-014, and NORI-017 to NORI-034 all inhibited the enzymatic activity of human MTP-2 in this assay. The IC50 values for the inhibition of human MTP-2 were in the range of approximately 0.083 nM to 17 nM. Table D. Figure 5.

[0319] In this cell-based assay, MTP-2 is expressed by the cells and is presented on the cell surface, where it serves as the site for the generation of activated MTP-2 that undergoes autoactivation and cleaves the substrate. Inhibitors in this assay can act by various molecular mechanisms. For example, an inhibitor that shows inhibition in this assay may bind to the MTP-2 enzyme precursor and inhibit its conversion to activated MTP-2 (Figure 2), thereby preventing the formation of the activated form of MTP-2 that would otherwise cleave the substrate in this assay, and / or the inhibitor in this assay may bind to the activated form of MTP-2 and inhibit it.

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

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

[0323] Refer to Example 5 for details of the preparation of the antigen reagent.

Example 5

[0324] Production of the antigen material Generation of constructs for protein expression To generate the purified proteins for use in the assays described herein, · The extracellular domain (ECD) aa77 - 855 of WT human matriptase-1 (uniprot accession number Q9Y5Y6) · Wild-type (WT) human MTP-2 (uniprot accession number Q8IU80) aa78 - 811 · DNA sequences encoding WT mouse MTP-2 (Uniprot accession number Q9DBI0) aa80-811 · DNA sequences encoding WT rat MTP-2 (NCBI accession number XP006242057.1) aa80-811 · DNA sequences encoding cynomolgus monkey MTP-2 (Uniprot accession number A0A2K5VAP0) aa73-800 were 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 leader sequence, codon-optimized for mammalian expression, and expressed.

[0327] To generate the MTP-2 antigen, expression can be significantly improved by co-expression with the tagless moHAI-2 ECD antigen (followed by purification of the His-tagged antigen via a nickel column). For this purpose, DNA encoding WT mouse HAI-2 (Uniprot accession number Q9WU03) aa28-197 was fused with an N-terminal immunoglobulin leader sequence, codon-optimized for mammalian expression, and expressed.

[0328] Using the golden gate method and AarI restriction sites, the DNA sequences were cloned into the pTT5 protein expression vector under the control of the CMV promoter. The expression plasmid was transfected into CHO-3E7 cells using the PEI transfection reagent.

[0329] Generation of full-length antigen constructs for stable cell line production For the purpose of screening MTP-2 specific antibodies, stable cell lines expressing the relevant antigens were generated.

[0330] The full-length DNA sequences encoding wild-type (WT) human MTP-2 (uniprot accession number Q8IU80) amino acids (aa) 1-811 and WT mouse MTP-2 (uniprot accession number Q9DBI0) aa1-811, each fused to an N-terminal eGFP and a C-terminal flag tag (DYKDDDDK), were codon-optimized for mammalian expression. This process was repeated for cynomolgus monkey MTP-2 (uniprot accession number A0A2K5VAP0) aa1-800 without tags. The DNA sequences were cloned into expression vectors under the control of a CMV promoter flanked by 3' and 5' piggyBac-specific terminal repeats to facilitate stable integration into the 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 vectors contained a puromycin selection cassette to facilitate the generation of stable cell lines.

[0331] Isoform 2 of WT human MTP-2 aa10-811 and WT mouse MTP-2 amino acids 13-811, i.e., the untagged form, was generated from the above constructs by PCR site-directed mutagenesis and recloned into the same expression vectors as before. Isoform 2 of the K253E, V736A, and K253E+V736A human WT MTP-2 (uniprot accession number Q8IU80) sequence aa10-811 variants, i.e., the untagged form, was also generated by PCR site-directed mutagenesis and recloned into the same expression vectors as before.

[0332] The full-length DNA sequences encoding WT human matriptase-1 (MTP-1) (uniprot accession number Q9Y5Y6) aa1-855 with a C-terminal His-tag fusion, WT human matriptase-3 (MTP-3) (uniprot accession number Q7RTY8) aa1-854 with a C-terminal His-tag fusion, and tagless WT mouse HAI-2 (uniprot accession number Q9WU03) aa1-252 were all codon-optimized for mammalian expression and cloned into the same expression vector with a CMV promoter as before. The expression vectors for MTP-1 and MTP-3 contained a puromycin selection cassette, and the expression vector for moHAI-2 contained a neomycin selection cassette that facilitated the generation of a double-stable cell line.

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

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

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

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

[0337] Twenty-four hours after transfection, the medium was supplemented with puromycin (2.5 μg / mL) or G418 (1 mg / mL), or both puromycin (2.5 μg / mL) and G418 (1 mg / mL), and 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. The expression of flag-tagged human or mouse MTP-2 constructs in cells was evaluated by flow cytometry using an anti-flag APC-conjugated antibody (Biolegend - 637308), and the 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-conjugated secondary antibody (Citeab - 115-605-071). After selection, stable tagless WT human MTP-2 and WT mouse MTP-2-expressing HEK293 cells, and stable tagless WT human MTP-2 and WT mouse MTP-2-expressing Hepa1-6 cells were FACS sorted for high expression. The expression of tagless human, mouse, and cynomolgus monkey MTP-2 constructs, including human and mouse mutants and human variant constructs, in cells was evaluated by flow cytometry using an APC-conjugated anti-MTP-2 antibody.

[0338] HEK293 and Hepa1-6 complete media were composed 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 suspension in CHO-S complete media composed of CD-CHO media 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 also be used.

Example 6

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

[0340] The affinity (K D ) for the antibodies tested was in the range of approximately 0.012 nM to 8.5 nM. Table K.

[0341]

Table 3

[0342] In addition, the above SPR method was used to evaluate the binding using the "headless" huMTP-2 ECD 78-576aa instead of the full ECD. The headless ECD corresponds to the MTP-2 ECD that does not have a serine protease domain. The amino acid sequence of the "headless" huMTP-2 ECD 78-576aa protein corresponds to the his-tag of the human matriptase-2 masked ECD protein shown in the sequence of Table S. For none of the seven antibodies was binding to the headless protein detected, suggesting that all epitopes of these antibodies are present in the serine protease domain.

[0343]

Table 4

[0344] NORI-011, NORI-008, NORI-003, and NORI-006 were all found to have cross-reactive binding to human, mouse, cynomolgus monkey, and rat MTP-2 ECD proteins. The sequences of these proteins used in SPR are given as the human matriptase-2 ECD protein his-tag, mouse matriptase-2 ECD protein his-tag, cynomolgus monkey matriptase-2 ECD protein his-tag, rat matriptase-2 ECD protein his-tag, and human matriptase-2 mask ECD protein his-tag, respectively, in the sequences of Table S. Binding to the human matriptase-2 mask ECD protein his-tag was not detected for any of these four antibodies, indicating that the binding is not to the his-tag and that the epitopes of all the antibodies are present in the serine protease domain. The true KD value of NORI-008 could not be accurately measured for either human MTP-2 or mouse MTP-2 because the dissociation rate was very slow.

Example 7

[0345] Binding to the MTP-2 Serine Protease Domain SPR analysis performed on the antibody panel in Example 6 showed that no binding to a truncated "headless" huMTP-2 protein lacking the serine protease domain was detected. 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 and preventing substrate cleavage.

[0346] HTRF binding assays of NORI-003, NORI-006, NORI-008, and NORI-011 were performed on this headless protein and confirmed that no detectable binding was present (Figure 6), while binding was detected for all human, mouse, and cynomolgus monkey MTP-2 proteins without C-terminal truncation (Figure 7). This also suggested that the binding epitopes of these inhibitory antibodies are present in the serine protease domain. Another antibody, namely NORI-036, bound to both the headless ECD and the full ECD, indicating that NORI-036 recognized the binding site of MTP-2 outside the serine protease domain.

Example 8

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

[0348]

Table 5

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

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

[0351] Inhibition of MTP-2 activity has been shown to be able to increase hepcidin expression by antibodies and thus prevent iron overload, the main cause of morbidity and mortality in beta-thalassemia. Therefore, antibodies that confirm such activity in vivo may be able to improve anemia in beta-thalassemia and be useful therapeutic agents for treating patients with iron overload or at risk of iron overload.

[0352] In vivo evaluation included assessing the increase in hepcidin gene mRNA transcription from liver cells and the decrease in serum iron in wild-type mice. The readout of this evaluation was obtained within 24 hours of dosing, allowing for 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 liver cells. The immediate effect of this inhibition is a 2- to 8-fold increase in hepcidin mRNA transcription that is observable within 6 hours of administration. The increased hepcidin expression results in the suppression of serum iron and thus transferrin saturation within an equivalent time frame. In healthy animals, continuous inhibition of MTP-2 and restriction of serum iron result in a decrease in mean corpuscular volume (MCV) and red blood cell distribution (RDW), which typically begins to manifest after 2 weeks if the drug is present and remains active.

[0354] The antibodies selected for in vivo testing were selected based on their exhibiting concentration-dependent inhibition of enzyme activity against human and mouse MTP-2 proteins and MTP-2 expressed on the cell surface. Antibodies were considered to have potential difficulties regarding expression and / or purification yield and to pose risks in terms of developability, and were excluded if in vitro, complete enzyme inhibition was not observed against either human ECD protein or mouse ECD protein, or if the generated IC50 values were considered to be very low.

[0355] Two antibodies that showed weak inhibition in the enzyme assay were also tested in vivo. Both had very poor in vivo function, which supported the hypothesis that an antibody must have a certain level of inhibition of MTP-2 enzyme activity to function well in vivo. Thus, in vitro inhibition is considered a criterion necessary for in vivo efficacy. However, perhaps this is not necessarily a sufficient condition for achieving an in vivo effect. For some antibodies, the inventors observed in vivo performance that was strong in the short term but not maintained over time. Other factors, such as pharmacokinetics or those related to anti-drug antibodies generated by the mouse, may affect the longer-term in vivo performance in these models.

Example 10

[0356] Protocol for Determining the Effect of Anti-MTP-2 Antibody on Hepcidin mRNA and Serum Iron in Wild-Type Mice Iron Assay Protocol To generate the data shown in Figures 13, 14, 15, 16, 17, and 18, iron quantification was performed using the QuantiChrom™ Iron Assay Kit (Bioassay System, DIFE-250).

[0357] Briefly, iron standards were prepared according to the kit protocol. Subsequently, 25 μl of the standard or sample was added to the wells of a 96-well plate, followed by the addition of 200 μl of Reagent A. The plate was then read at 595 nm in a microplate spectrophotometer (reading A). Next, 10 μl of Reagent B was added to the wells, followed by the addition of 10 μl of Reagent C. The plate was then incubated at room temperature for 40 minutes and read at 595 nm in a plate reader (reading B). For all wells, the increase in absorbance was calculated by subtracting reading A from reading B. A 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 measurement (Ferene) kit according to the protocol supplied in the kit.

[0359] Briefly, 25 μl of the standard or serum from the study animals was incubated in a 96-well with 125 μL of Solution R1 and 25 μL of Solution R2, and then the plate was read at 600 nm CLF protocol in an envision microplate reader (absorbance A1). 2.5 μl of chromogen was added. After a 20-minute incubation at room temperature, the plate was read based on the same envision protocol as before (absorbance A2). The results were calculated by:

Equation

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

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

Number

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

[0363] Analysis of Serum Antibody Levels The following assay was used to confirm the antibody concentration in serum: 96-well plates were coated with 50 μL / well of mouse anti-human IgG4 Fc (2 μg / mL in PBS) overnight at 4 °C. The plates were washed three times with 300 μL / well of PBD-T (PBS and 0.1% Tween) using a plate washer. The plates were blocked for 1 hour at room temperature using PBS with 1% BSA added. Samples were diluted (using stored mouse serum), and QC and standard curves (10 steps (7.81 - 2000 ng / ml)) were prepared. Next, the plates were washed three times with 300 μL / well of PBD-T (PBS and 0.1% Tween) using a plate washer. 50 μL of the standard curve, samples, or QC per well was added to the assay plate, and then the assay plate was incubated for 1 hour at room temperature with shaking at 300 RPM. Next, the plates were washed three times with 300 μL / well of PBD-T (PBS and 0.1% Tween) using a plate washer. 50 μL / well of HRP-conjugated mouse anti-human kappa diluted 1 / 12000 in PBS with 1% BSA added was added to the plates, and then the plates were incubated for 1 hour at RT with shaking at 300 RPM. Next, the plates were 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. Incubation was carried out for 10 minutes at RT in the dark. Then, 100 μL / well of stop solution (1 M sulfuric acid) was added. The optical density of each well was measured using a microplate reader set at 450 nm with a reference reading above 540 nm. The reference reading value was subtracted from the reading value at 450 nm. Next, the data was imported into Softmax Pro, and regression was performed using 4PL curve fitting with a weighting factor of 1 / y for the standard substances of the sample concentrations read from the standard curve.

[0364] RNA extraction The Qiagen RNeasy Plus Mini Kit was used to produce RNA from liver samples according to the kit protocol. Frozen samples were thawed on wet ice. Then, 600 μl of Buffer RLT Plus was added to the samples. Next, a plastic pestle was used to homogenize the samples. Then, the samples were triturated using a 1 ml syringe and a 20G needle. The samples were then centrifuged, and the supernatant was placed into a gDNA elimination spin column in a 2 ml Eppendorf, discarding the pellet. The spin column and tube were centrifuged, retaining the flow-through and discarding the column. Then, 600 μl of 70% ethanol was added to the flow-through. Then, 700 μl of the sample was loaded onto an RNeasy Spin Column placed in a 2 ml collection tube and then centrifuged. The flow-through was discarded. Then, the spin column was returned to the 2 ml collection tube. 700 μl of Buffer RW1 was added to the spin column. Then, the spin column was centrifuged, discarding the liquid flow-through. The spin column was returned to the 2 ml collection tube, 500 μl of Buffer RPE was added to the spin column, and the spin column was centrifuged. The flow-through was discarded, and the washing of the column with Buffer RPE was repeated as above. Centrifugation was performed at 8000×g for 15 seconds. The spin column was placed into a new 2 ml collection tube and centrifuged for up to 1 minute to dry the membrane. The spin column was placed into a new 1.5 ml collection tube, and 30 - 50 μl of RNase-free water was added directly to the spin column membrane. The spin column was centrifuged for 1 minute to elute the RNA. The RNA levels in the flow-through were estimated using a nanodrop (see below), and then the solution was 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 levels of mouse hepcidin (hamp) mRNA were measured by qRT-PCR and normalized to the mouse hypoxanthine-guanine phosphoribosyltransferase (HPRT) mRNA housekeeping gene. Then, 5 μl of the mRNA extract (25 ng in total) was mixed with 10 μl of the QuantiTect probe RT-PCR kit, 1 μl of the 20X hamp FAM probe mix, 1 μl of the 20X HPRT VIC probe mix, 0.5 μl of the 40X taqman RT enzyme mix, and 2.5 μl of RNA-free H2O to make a final volume of 20 μl in a 96-well semi-skirted qRT-PCR plate. The settings for the qRT-PCR reaction included a reverse transcription step at 48 °C for 15 minutes, followed by an activation step at 95 °C for 10 minutes, and then 40 cycles of 95 °C for 15 seconds and 60 °C for 1 minute. Then, the -ΔCt value was calculated by subtracting the Ct value of Hamp from that of HPRT.

Example 11

[0366] Single-dose evaluation over 24 hours of four IgG / lambda anti-MTP2 antibodies in normal mice regarding reducing serum iron and transferrin saturation NORI-009, NORI-010, NORI-012, and NORI-034 were included in the first evaluation in healthy mice as fully human IgG4λ mAbs. One intraperitoneal injection antibody was administered to 9-week-old C57BL / 6 male mice at 10 mg / kg (150 μl / mouse) and 5 mice per group. The 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 gram of body weight) and sacrificed 4 hours later. LPS (lipopolysaccharide derived from Escherichia coli) mimics bacterial infection, and mice respond by inducing hepcidin expression, reducing serum iron. LPS serves as a positive control for drugs known to cause an acute inflammatory response and based on the inflammatory response, cause the largest increase in hepcidin. Real-time PCR of hamp, Id1, Atho8, SMAD7, CRP, and Saa3 mRNA was performed on liver tissue. Hematological parameters were also determined. The number of red blood cells was determined and hemoglobin was measured. Serum iron from all mice was determined and transferrin saturation was calculated.

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

[0368] This experiment demonstrated the biological relevance between MTP-2 and the BMP / SMAD / hepcidin pathway and for the first time showed that serum iron can be decreased in normal mice by antibody-mediated inhibition. Since NORI-034 was considered inactive in vivo, this antibody was not investigated further.

Example 12

[0369] Time-course evaluation of NORI-010 after single ip injection into normal mice Antibody NORI-010 is one of the antibodies that showed a favorable profile in Example 11, and therefore it was of interest to determine the effects observed over time for that antibody. One ip injection antibody was administered to C57BL / 6 male mice at 10 mg / kg (150 μl / mouse) and 5 mice per group. The groups were 5 mice sacrificed at 24 hours, 72 hours, 1 week, and 2 weeks. The analysis was the same as in Example 11 but included hematocrit (HCT), mean corpuscular hemoglobin (MCH), and red blood cell distribution width (RDW), a measure of cell size related to red blood cell volume.

[0370] With administration of the antibody at 10 mg / kg, the hepcidin increase was maintained for 2 weeks, and serum iron and TSAT decreased over that duration. The MCV value decreased due to consistent iron restriction. Figure 10.

[0371] With administration of the antibody at 3 mg / kg, the effect on the hepcidin increase was lost by 2 weeks due to the lower dose. Nevertheless, serum iron and TSAT remained decreased over 2 weeks, and the MCV also decreased as in the case of the 10 mg / kg dose. Figure 11.

Example 13

[0372] 24-hour evaluation of two IgG / kappa anti-MTP2 antibodies and two IgG / lambda anti-MTP-2 antibodies regarding reduction of serum iron and transferrin saturation NORI-008 of mouse IgG1 (moIgG1) construct 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 of moIgG1 and huIgG4PE constructs, respectively. The latter control antibodies were cross-reactive binders to MTP-2 but did not inhibit the enzyme activity of human ECD protein or mouse ECD protein in vitro.

[0373] Nine-week-old C57BL / 6 male mice were administered one intraperitoneal injection antibody at 10 mg / kg (150 μl / mouse) and 3 - 5 mice per group. The 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. The number of red blood cells was determined and hemoglobin was measured. Serum iron from all mice was determined and transferrin saturation was calculated.

[0374] Here, the inventors have 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 the 24-hour time point. Figure 12.

[0375] This demonstrates that both antibodies maximally inhibit MTP-2 activity in vivo. NORI-036 and NORI-037 are binders to MTP-2 but have no effect on hepcidin expression or serum iron in vivo. This demonstrates that the in vitro screening strategy has been successful 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 injection at 10, 3, and 1 mg / kg into normal mice This experiment shows the dose-time relationship after ip injection of the anti-MTP-2 antibody NORI-008 by the change in serum iron concentration.

[0377] Male C57BL6J mice, 22 - 28 g, n = 5 / group, were administered a single ip injection of 2, 20, or 200 μg of the fully human IgG4 anti-MTP-2 antibody NORI-008. One group was administered a 200 μg dose of a human IgG4 isotype control antibody and sorted 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 sorted at a separate dose. Blood was collected from animals in one group treated with each dose on days 1, 7, and 14, and serum iron concentrations were confirmed to evaluate the efficacy of NORI-008.

[0378] Samples from all doses and time points were then analyzed for hepcidin expression in the liver and antibody levels in serum at day 7 (PK analysis). Antibodies were undetectable at any time point in the 2 μg group, and only 3 out of 5 had measurable levels 24 hours after the 20 μg dose.

[0379] At the 200 μg dose, the NORI-008 effect lasted at least one week in vivo, after which hepcidin expression and serum iron returned to normal at the 2-week time point. At the 20 μg dose, the effect lasted at least one day, after which it returned to normal at the 1-week time point. In the case of the 2 μg dose, no effect was observed. The duration of the effect correlates with the PK value of the antibody concentration. That is, when the antibody drops below the critical concentration, the effect on MTP-2 inhibition is lost, and hepcidin expression and serum iron return to normal. Figure 13.

Example 15

[0380] In vivo evaluation at 1-week time point of six IgG4 / kappa anti-MTP2 antibodies in normal mice regarding reducing serum iron and transferrin saturation This study evaluated six fully human IgG4 anti-MTP-2 antibodies for their ability to reduce serum iron concentration after ip injection.

[0381] Male C57BL6J mice weighing 25 - 32 g and n = 3 - 5 / group were administered a single ip injection of 10 mg / kg of a fully human IgG4 anti-MTP-2 antibody. Eight groups were included in the study, and each animal was administered a single ip dose of 10 mg / kg of one of seven fully human IgG4PE anti-MTP2 antibodies, namely NORI-005, NORI-004, NORI-002, NORI-001, NORI-007, NORI-006, and NORI-010, or a human IgG4 isotype control antibody. NORI-010 was added to this experiment to compare with the remaining antibodies used in this experiment as a positive control and benchmark anti-MTP-2 antibody. All groups contained 5 animals except for NORI-005 (n = 3) and NORI-002 (n = 4). Blood from the animals was collected on day 7, and the serum iron concentration was confirmed to evaluate iron parameters, hamp mRNA, and the IgG level of the anti-MTP-2 antibody.

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

Example 16

[0383] In vivo evaluation at 1 week of two IgG / kappa anti-MTP2 antibodies and one IgG / lambda anti-MTP2 antibody (10 mg / kg I.P. dose) of both huIgG4 and moIgG1 skeletons regarding reducing serum iron This study evaluated the ability of three anti-MTP-2 antibodies of fully human IgG4 or mouse IgG1 composition to reduce serum iron concentration after subcutaneous injection.

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

[0385] Figure 15.

Example 17

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

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

[0388] Blood was collected from the animals on day 7, and the serum iron concentration and IgG level were confirmed. Figure 16.

[0389] The results of this experiment showed that for both the ip and sc dosing methods, the concentration of the antibody in the serum was equivalent at the 1 - week time point. As a result, the serum iron reduction was also equivalent for both dosing methods and decreased due to MTP - 2 inhibition as previously seen.

Example 18

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

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

[0392] Blood was collected from the animals on days 1, 3, 7, 9, 14, and 21. Serum iron and serum antibody concentrations were measured at all time points.

[0393] NORI-010 produced a pharmacological effect at 10 mg / kg, decreasing serum iron until day 9 and then returning to normal, but the effect lasted only 24 hours at 3 mg / kg. NORI-008 produced an effect at 10 mg / kg, decreasing serum iron until day 9 and then returning to normal, but the effect lasted only 72 hours at 3 mg / kg. The PD of these antibodies was well correlated with serum IgG levels, and the iron-lowering effect was lost as the antibody concentration decreased. Figure 17.

Example 19

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

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

[0396] Blood was collected from the animals on days 1, 3, 7, 9, 14, and 21. Serum iron and serum antibody concentrations were measured at all time points.

[0397] NORI-008 and NORI-010 also showed an effect with a duration similar to that of Example 18 in this case. The effect on reducing serum iron lasted until day 9 and then returned to normal. Both NORI-003 and NORI-006 showed a more favorable duration effect, and serum iron remained suppressed throughout the 21-day study.

[0398] PK analysis of this study showed a significant difference in Cmax of the four antibodies, with NORI-003 and NORI-006 having much higher serum antibody concentrations compared to NORI-008 and NORI-010 on day 3. As a result, these two antibodies showed a more favorable PK profile and remained above the critical serum antibody concentration for inhibiting MTP-2 for a longer period. Therefore, the effect of suppressing serum iron persisted. Figure 18.

Example 20

[0399] In vivo evaluation in a mouse model of beta-thalassemia after single intraperitoneal 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 aging, with Hb levels between 7-9 g / dL, abnormal erythrocyte morphology, increased reticulocyte counts, ineffective and extramedullary erythropoiesis, hepatosplenomegaly, and iron overload in the liver and spleen, similar to that of beta-thalassemia intermedia in humans. According to the activity of NORI-010 observed in Examples 18-20, 10 mg / kg of this antibody was injected ip, and the animals were sacrificed 2 weeks after this single dose (n = 3 per time point). In addition, one group of animals was analyzed for hepcidin mRNA 24 hours later. A human IgG4 isotype control antibody was used at the same dose as a negative control.

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

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

Example 21

[0402] Evaluation of 8-week repeated administration of treatment with NORI-011-M in a murine model of beta-thalassemia with and without concurrent treatment with erythropoietin The purpose of this study was to examine the effect on hematological parameters after a treatment period longer than that in Example 20. For this purpose, Hbbth3 / + mice (n = 5 / group) were intraperitoneally injected with 10 mg / kg of the NORI-011-M antibody once a week for 8 weeks. For this longer-term repeated administration experiment, the isotype of NORI-011 was reconfigured with respect to the murine constant regions (murine IgG1 and murine lambda constant regions) to avoid or suppress immunogenicity and the production of anti-MTP2 antibodies in mice. In connection with this reconfiguration, minor modifications were 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 murine isotype IgG1.

[0403] In addition, this study attempted to investigate the effect of concurrent administration of erythropoietin (epo), which is known to improve some important parameters but has a harmful effect on spleen size due to iron loading and excessive cell death. The favorable effect of this combination was confirmed in an experiment in Hbbth3 / + mice in which epo was supplied by overexpressing recombinant fibroblasts and reduction of matriptase-2 activity was supplied 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 co-administered with NORI-011-M at 30 μg / kg once a week to one group of this study. As a control, epo was also co-administered with an isotype control antibody to present the effect of epo alone on the readout of this study. Figure 20 shows the results regarding individual hematological parameters and hepcidin mRNA levels in liver samples.

[0404] The results also showed that in this case, hepcidin levels were consistently maintained at elevated levels over the course of the 8-week experiment (a). Thus, as seen in the above examples, this is expected to result in iron restriction and subsequent reduction of serum iron and liver iron content (b), as well as reduction of MCV (f). The red blood cell count and hemoglobin did not substantially increase with NORI-011-M alone. However, erythrocyte maturation was improved in the spleen, and the proportion of stage V (mature) cells was much larger than in any other group (i). The increase in spleen weight was also approximately halved in the case of dosing with NORI-011-M alone, indicating that the restriction of iron supply caused by the NORI-011-M blocker has a normalizing effect on erythrocyte maturation (k).

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

[0406] The combined treatment of NORI-011-M and epo brought about a combination of favorable effects. This was a slightly reduced effect on the red blood cell count (c) and hemoglobin (d) compared to epo alone, but overall was represented by a more balanced therapeutic effect compared to epo alone, including a continued reduction of hepatic iron overload (b) and a decrease in splenomegaly (k). It is thought that these synergistic effects in the clinical setting can be optimized by optimizing the treatment ratio of matriptase inhibition and epo stimulation.

Example 22

[0407] 8-week repeated administration evaluation of treatment with NORI-011-M in a mouse model of beta-thalassemia with and without concurrent treatment with ActRIIB-Fc fusion protein In this study, we demonstrate that concurrent 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 and ActRIIB-Fc alone. This synergistic effect may reflect the different mechanisms of action of these two drugs, where ActRIIB-Fc promotes the maturation of erythroid precursors during erythropoiesis, while NORI-11-M causes a slowdown in erythropoiesis that results in iron restriction and normalization, and thus more efficient production of more mature erythrocytes.

[0408] The work in this study was performed over 8 weeks with multiple administrations of both drugs using the methods and procedures outlined generally in Example 21 above. As before, Hbbth3 / + mice (n = 5 / group) were intraperitoneally injected with 10 mg / kg of NORI-011-M antibody once a week for 8 weeks. ActRIIB-Fc 10 mg / kg was intraperitoneally injected twice a week for 8 weeks. The twice as many administrations of ActRIIb-Fc compared to the antibody reflected a previously published dosing protocol for a similar activin receptor ligand trap molecule (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 a modified human ActRIIB extracellular domain (uniprot - Q13705, residues 26 - 131) with an L79D modification, expressed in floating CHO cells, purified via the Fc domain, and fused to the mouse IgG2a-Fc domain (uniprot - P01863, residues 99 - 330) via a short 3x glycine linker.

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

[0411] Figure 21 shows the results regarding individual hematological parameters and hepcidin mRNA levels in liver samples. Consistent with Example 21 above, the results also show that 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 liver iron levels (Figure 21b), a decrease in serum iron, and a reduction in mean corpuscular volume (MCV) (Figure 21f). In the case of treatment with ActRIIB-Fc alone, no increase in hepcidin (hamp) mRNA was observed, and co-treatment resulted in the same outcome as when using NORI-11-M alone. Thus, despite a greater number of dosing times, ActRIIB-Fc was unable to directly increase hepcidin expression or correspondingly reduce liver iron levels.

[0412] Mean corpuscular hemoglobin (MCH) was reduced in mice treated with NORI-11-M, reflecting iron supply restriction, but was also reduced by ActRIIB-Fc (Figure 21h), which reflected that a greater number of cells were produced (indicated by increases in total red blood cell count and hemoglobin), and thus less hemoglobin was available per cell. Mean corpuscular volume (MCV) was also reduced (Figure 21f). Usually, low MCV indicates microcytic anemia, but in this case, iron restriction (caused by the antibody) or hemoglobin deficiency (caused by enhanced maturation of more RBCs due to ActRIIB-Fc) causes a reduction in availability per cell, as already shown by the MCH results.

[0413] All treatment groups improved the "quality" of the generated red blood cells, as reflected by a decrease (normalization) in red blood cell distribution width (RDW) (Figure 21g). Thus, the uniformity of red blood cells was improved. In the case of 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 further increases beyond treatment with ActRIIB-Fc alone, indicating that the maturation effect on erythropoiesis 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. The red blood cell count recovered to wild-type levels, and both hematocrit levels and hemoglobin levels were significantly improved by treatment with ActRIIB-Fc alone or in combination with NORI-11-M. This is important to show that the iron reduction caused by NORI-11-M does not cancel out the beneficial effect on erythropoiesis caused by ActRIIB-Fc. Thus, overall, the advantages of both treatments can still be achieved even when they are used in combination, and it is possible to both reduce iron overload and increase red blood cell production / maturation.

[0414] Furthermore, when the two agents are actually used together, they provide a greater overall benefit due to a therapeutic ability that exceeds their use alone, as shown by the effect on reducing splenomegaly, for example.

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

[0416] The inventors reported in Example 21 that the use of EPO had a favorable effect on the number of erythrocytes (Figure 20c) and total hemoglobin (Figure 20d), as expected for a stimulator of erythropoiesis. However, as is already known in the medical field, the stimulation of increased erythropoiesis by the use of EPO causes an increase in cell death and apoptosis, as well as a further increase in the associated spleen size and weight, so-called splenomegaly, in the context of continued beta-globin synthesis deficiency (Figure 20k). In this regard, in this example, the use of NORI-11-M or ActRIIB-Fc did not cause an increase in spleen weight compared to untreated animals (Figure 21k). Furthermore, although not to the level of healthy animals, a significant reduction in spleen weight was actually present. The combined treatment of NORI-011-M and ActRIIB-Fc was considered to be far superior to monotherapy alone by further reducing spleen weight.

[0417] Sequence 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 are embodiments of the present invention.

[0418] [Table 6-1]

Table 6-2

Table 6-3

Table 6-4

Table 6-5

Table 6-6

Table 6-7

Table 6-8

Table 6-9

Table 6-10

Table 6-11

Table 6-12

Table 6-13

Table 6-14

Table 6-15

Table 6-16

Table 6-17

Table 6-18

Table 6-19

Table 6-20

Table 6-21

Table 6-22

Table 6-23

Table 6-24

Table 6-25

Table 6-26

Table 6-27

Table 6-28

Table 6-29

Table 6-30

Table 6-31

Table 6-32

Table 6-33

Table 6-34

Table 6-35

Table 6-36

Table 6-37

Table 6-38

Table 6-39

Table 6-40

Table 6-41

Table 6-42

Table 6-43

Table 6-44

Table 6-45

Table 6-46

Table 6-47

Table 6-48

Table 6-49

Table 6-50

Table 6-51

Table 6-52

Table 6-53

Table 6-54

Table 6-55

Table 6-56

Table 6-57

Table 6-58

Table 6-59

Table 6-60

Table 6-61

Table 6-62

Table 6-63

Table 6-64

Table 6-65

Table 6-66

Table 6-67

Table 6-68

Table 6-69

Table 6-70

Table 6-71

Table 6-72

Table 6-73

Table 6-74

Table 6-75

Table 6-76

Table 6-77

Table 6-78

Table 6-79

Table 6-80

Table 6-81

Table 6-82

Table 6-83

Table 6-84

Table 6-85

Table 6-86

Table 6-87

Table 6-88

Table 6-89

Table 6-90

Table 6-91

Table 6-92

Table 6-93

Table 6-94

Table 6-95

Table 6-96

Table 6-97

Table 6-98

Table 6-99

Table 6-100

Table 6-101

Table 6-102

Table 6-103

Table 6-104

Table 6-105

Table 6-106

Table 6-107

Table 6-108

Table 6-109

Table 6-110

Claims

1. A binder polypeptide that binds to human MTP-2, comprising: (a) A heavy chain variable (VH) domain comprising heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 48, HCDR2 comprises the amino acid sequence of SEQ ID NO: 49, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 50; and (b) A light chain variable (VL) domain comprising light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence of SEQ ID NO: 58, LCDR2 comprises the amino acid sequence of SEQ ID NO: 59, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 60 A binder polypeptide comprising the above.

2. (a) HCDR1 consists of the amino acid sequence of SEQ ID NO: 48, HCDR2 consists of the amino acid sequence of SEQ ID NO: 49, and HCDR3 consists of the amino acid sequence of SEQ ID NO: 50; (b) LCDR1 consists of the amino acid sequence of SEQ ID NO: 58, LCDR2 consists of the amino acid sequence of SEQ ID NO: 59, and LCDR3 consists of the amino acid sequence of SEQ ID NO: 60, The binder polypeptide according to Claim 1.

3. (a) The VH domain comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 67; (b) The VL domain comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 63, The binder polypeptide according to Claim 1 or 2.

4. (a) The VH domain comprises the amino acid sequence of SEQ ID NO: 67; (b) The VL domain comprises the amino acid sequence of SEQ ID NO: 63, The binder polypeptide according to any one of Claims 1 to 3.

5. (a) The VH domain consists of the amino acid sequence of SEQ ID NO: 67; (b) The VL domain consists of the amino acid sequence of SEQ ID NO: 63, The binder polypeptide according to any one of Claims 1 to 4.

6. The binder polypeptide according to any one of Claims 1 to 5, further comprising an antibody constant region.

7. The binder polypeptide according to any one of Claims 1 to 6, further comprising an IgG antibody.

8. The binder polypeptide according to any one of Claims 1 to 7, further comprising a human IgG4 Fc constant region.

9. (a) A heavy chain comprising the amino acid sequence of SEQ ID NO: 69, and (b) A light chain comprising the amino acid sequence of SEQ ID NO: 65 The binder polypeptide according to any one of claims 1 to 8, comprising

10. (a) a heavy chain consisting of the amino acid sequence of SEQ ID NO: 69, and (b) a light chain consisting of the amino acid sequence of SEQ ID NO: 65 The binder polypeptide according to any one of claims 1 to 9, comprising

11. A nucleic acid encoding the binder polypeptide according to any one of claims 1 to 10.

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

13. A composition comprising (a) the binder polypeptide according to any one of claims 1 to 10, and (b) at least one pharmaceutically acceptable excipient.

14. The composition according to claim 13, for reducing the absorption of dietary iron in a patient.

15. The composition according to claim 13, for treating iron overload in a patient.

16. The composition according to claim 13, for increasing the expression of hepcidin from hepatocytes in a patient.

17. The composition according to claim 13, for reducing anemia caused by iron overload in a patient.

18. The composition according to claim 13, for reducing the serum iron concentration in a patient.

19. The composition according to claim 13, for reducing the iron saturation of transferrin in a patient.

20. The patient has beta-thalassemia, myelodysplastic syndrome, polycythemia vera, hemochromatosis, or refractory anemia with ring sideroblasts (RARS). The composition according to any one of claims 14 to 19.

21. A composition comprising (i) the binder polypeptide according to any one of claims 1 to 10, and (ii) an antagonist of a TGFβ superfamily ligand.

22. The TGFβ superfamily ligand is selected from activin, GDF-11, and bone morphogenetic protein. The composition according to claim 21.

23. The composition according to claim 21 or 22, for treating iron overload and normalizing erythropoiesis in a patient.

24. A composition comprising (i) the binder polypeptide according to any one of claims 1 to 10, and (ii) erythropoietin.

25. The composition according to claim 24, for treating anemia associated with iron overload in a patient.

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