Anti-TMPRSS6 antibodies and uses thereof

Abstract

Provided are antibodies and antigen-binding fragments thereof that bind to type II transmembrane serine protease 6 (TMPRSS6) on the cell surface and increase hepcidin expression, as well as methods for treating iron metabolism disorders and myeloproliferative disorders using anti-TMPRSS6 antibodies and fragments thereof.

Description

[Technical Field]

[0001] The present disclosure relates to antibodies and antigen-binding fragments that bind to TMPRSS6, and to treating disorders including iron metabolism disorders and myeloproliferative neoplasms using antibodies and antigen-binding fragments that bind to TMPRSS6. [Background technology]

[0002] Type II transmembrane serine protease 6 (TMPRSS6) is encoded by the TMPRSS6 gene and is primarily expressed in the liver. TMPRSS6 contains a type II transmembrane domain followed by a sea urchin sperm protein, enteropeptidase, and agrin (SEA) domain, a stem region containing two complement factors C1r / C1s, a sea urchin embryonic growth factor and bone morphogenetic protein (CUB) domain, three low-density lipoprotein receptor (LDLR) class A repeats, and a C-terminal trypsin-like serine protease domain (Wang, C.-Y. et al., Front. Pharmacol. 2014. 5:114). Other names for TMPRSS6 (EC 3.4.21) include matriptase-2, transmembrane protease serine 6, membrane-bound mosaic serine protease matriptase-2, and MT2.

[0003] TMPRSS6 plays a critical role in iron homeostasis through the BMP-SMAD signaling pathway, which regulates the expression of hepcidin, a hormone that controls iron absorption and mobilization from iron stores. Hepcidin (also known as HAMP (hepcidin antimicrobial protein or peptide) in humans and non-human primates and HAMP in mice and rats) regulates systemic iron homeostasis by controlling the functional activity of ferroportin, the only iron efflux channel. Hepcidin reduces plasma iron levels by binding to ferroportin and causing the internalization and degradation of the complex, thereby preventing iron absorption in the small intestine and the release of iron stores. Chronically elevated hepcidin levels cause systemic iron deficiency, while hepcidin deficiency causes systemic iron overload.

[0004] TMPRSS6 negatively regulates hepcidin production through a transmembrane signaling pathway that is induced by iron deficiency and inhibits HAMP activation (Du, X. et al., Science 2008. 320: 1088-1092; Wang, C.-Y. et al., Front. Pharmacol. 2014. 5:114). Low blood iron levels induce this pathway, reducing hepcidin production and allowing more dietary iron to be absorbed from the intestine and transported from storage sites into the bloodstream. In acute iron-deficient rats, hepatic TMPRSS6 protein levels are upregulated, suppressing hepcidin expression and production (Wang, C.-Y. et al., Front. Pharmacol. 2014. 5:114). Mutations in the entire TMPRSS6 molecule, particularly the extracellular domain, have been identified in subjects with iron deficiency anemia, particularly iron-refractory iron deficiency anemia (IRIDA), which does not respond to oral iron treatment and only partially responds to parenteral iron therapy (Wang, C.-Y. et al., Front.Pharmacol.2014.5:114). Loss-of-function mutations in TMPRSS6 in humans result in elevated hepcidin levels and iron deficiency anemia (Camaschella, C., N Engl Journal Med 2013.168:24), because overproduction of hepcidin leads to defective iron absorption and utilization.

[0005] Iron overload occurs when excess iron accumulates in tissues and organs, disrupting their normal function. Iron toxicity is a common complication of iron overload and results in high mortality rates as a result of iron accumulation in major organs. β-thalassemia is an iron overload disorder that occurs when mutations in the HBB gene result in reduced or absent β-globin (beta-globin) production, leading to erythroblast apoptosis and a lack of mature red blood cells, resulting in ineffective erythropoiesis, which causes excessive iron absorption leading to anemia and iron toxicity. In patients with β-thalassemia, hepcidin is abnormally suppressed in relation to the patient's iron-loading status, resulting in hepcidin deficiency and consequent excessive iron absorption and systemic iron overload. Ineffective erythropoiesis in other disorders, such as myelodysplastic syndromes (MDS), dyserythropoietic anemia, and sideroblastic anemia, is similarly characterized by hepcidin deficiency, leading to iron overload. Hemochromatosis, such as hemochromatosis type 1 or hereditary hemochromatosis, is an iron overload disorder characterized by intestinal hyperabsorption of dietary iron and a pathological increase in total body iron stores. Current standard therapies for treating iron overload include blood transfusions for ineffective erythropoiesis, iron chelation therapy, which can further exacerbate iron overload, and phlebotomy or splenectomy to manage symptoms. Therapeutic approaches currently under development include gene therapy targeting the HBB gene, gene therapy and gene editing targeting other related genes, hepcidin mimetics, Fc fusion proteins targeting TGF superfamily ligands to inhibit SMAD signaling, antisense RNA drugs targeting TMPRSS6 (e.g., El-Beshlawy A., et al., Blood Cells, Molecules and Diseases 2019.76:53-58), and iRNA drugs targeting TMPRSS6.

[0006] Polycythemia vera (PV) is a chronic myeloproliferative neoplasm in which the JAK2 / STAT5 signaling pathway is constitutively activated, resulting in increased red blood cell mass and erythroid hyperplasia. The primary cause of death is thrombotic complications due to increased blood viscosity. Possible downstream conditions of constitutively activated JAK2 / STAT5 signaling include the simultaneous occurrence of abnormal erythropoiesis, an inflammatory environment, decreased systemic iron concentrations, and altered hypoxic responsiveness, which may directly affect iron absorption in certain tissues. Any or all of these may be involved in iron metabolism in PV. (Ginzburg, YZ et al., Leukemia 2018.32:2105-2116) Evidence suggests that systemic iron deficiency or red blood cell-targeted iron restriction may be beneficial in reducing erythrocytosis and normalizing hematocrit in PV. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention relates to novel antibodies and antigen-binding fragments thereof that bind to TMPRSS6, and methods of making and using antibodies and antigen-binding fragments thereof that bind to TMPRSS6. [Means for solving the problem]

[0008] The present disclosure provides anti-TMPRSS6 antibodies, nucleic acids encoding anti-TMPRSS6 antibodies, and methods for making and using anti-TMPRSS6 antibodies. The anti-TMPRSS6 antibodies disclosed herein include anti-TMPRSS6 antibodies and fragments thereof capable of binding to TMPRSS6. The anti-TMPRSS6 antibodies disclosed herein are capable of binding to human TMPRSS6 on the surface of cells expressing human TMPRSS6. The present disclosure provides anti-TMPRSS6 antibodies for therapeutic and diagnostic use. The anti-TMPRSS6 antibodies disclosed herein can be used to treat disorders of iron metabolism, such as iron overload, particularly β-thalassemia, including but not limited to non-transfusion-dependent thalassemia, and other disorders of ineffective erythropoiesis. The anti-TMPRSS6 antibodies disclosed herein can be used to treat myeloproliferative disorders, such as polycythemia vera (PV), which is characterized by erythrocytosis and erythroid hyperplasia.

[0009] In one aspect, an anti-TMPRSS6 antibody is provided that can bind to TMPRSS6 on the surface of a cell that expresses TMPRSS6 and modulate the activity of at least one component involved in iron metabolism (wherein the component may be a molecule or a biological process related to the function of TMPRSS6). In certain embodiments, the anti-TMPRSS6 antibody disclosed herein can modulate the activity of at least one component involved in the regulation of hepcidin expression. In certain embodiments, the anti-TMPRSS6 antibody disclosed herein can substantially inhibit TMPRSS6 repression of hepcidin expression. In certain embodiments, the anti-TMPRSS6 antibody disclosed herein can increase hepcidin expression. In certain embodiments, the anti-TMPRSS6 antibody disclosed herein can increase hepcidin promoter activity. In certain embodiments, the anti-TMPRSS6 antibody disclosed herein can substantially inhibit TMPRSS6 repression of BMP / SMAD pathway-induced hepcidin expression. The anti-TMPRSS6 antibodies disclosed herein can modulate hepcidin expression, including, but not limited to, substantially inhibiting TMPRSS6 repression of hepcidin expression, increasing hepcidin expression, increasing hepcidin promoter activity, or substantially inhibiting TMPRSS6 repression of hepcidin expression induced by the BMP / SMAD pathway, in a dose-dependent manner. In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein can modulate hepcidin expression in a dose-dependent manner. In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein can increase serum hepcidin levels in a dose-dependent manner when administered to a subject. In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein can reduce serum iron levels in a dose-dependent manner when administered to a subject. In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein can increase liver hepcidin RNA levels in a dose-dependent manner when administered to a subject.In certain embodiments, when the anti-TMPRSS6 antibody disclosed herein is administered to a subject known or suspected of having iron overload, particularly β-thalassemia, it can reduce liver non-heme iron, increase serum hepcidin, increase liver hepcidin RNA, reduce splenomegaly, increase red blood cell count (RBC), increase hematocrit (HCT), reduce red blood cell distribution width (RDW), and increase the production of mature red blood cells (increased erythropoiesis). In certain embodiments, when the anti-TMPRSS6 antibody disclosed herein is administered to a subject known or suspected of having a myeloproliferative disorder, such as a myeloproliferative neoplasm, particularly polycythemia vera (PV), it can reduce RBC, reduce HCT, reduce hemoglobin (HGB), reduce mean corpuscular volume (MCV), and reduce RDW.

[0010] In another aspect, the anti-TMPRSS6 antibodies disclosed herein exhibit cross-reactivity with at least one non-human TMPRSS6. In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein are capable of binding to at least one non-human TMPRSS6 on the surface of a cell expressing at least one non-human TMPRSS6. The anti-TMPRSS6 antibodies disclosed herein may be capable of binding to human TMPRSS6 and mouse TMPRSS6. The anti-TMPRSS6 antibodies disclosed herein may be capable of binding to human TMPRSS6 and cynomolgus monkey TMPRSS6. The anti-TMPRSS6 antibodies disclosed herein may be capable of binding to each of human TMPRSS6, mouse TMPRSS6, and cynomolgus monkey TMPRSS6.

[0011] In another aspect, the anti-TMPRSS6 antibodies disclosed herein specifically bind to TMPRSS6 (matriptase-2). In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein bind to TMPRSS6 (matriptase-2) and do not exhibit detectable binding to matriptase homologs. In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein bind to human TMPRSS6 (matriptase-2) and do not exhibit detectable binding to human matriptase-1 (ST14). In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein bind to human TMPRSS6 (matriptase-2) and do not exhibit detectable binding to human matriptase-3 (TMPRSS7). In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein bind to human TMPRSS6 (matriptase-2) and do not exhibit detectable binding to either human matriptase-1 (ST14) or human matriptase-3 (TMPRSS7).

[0012] The anti-TMPRSS6 antibodies disclosed herein may be monoclonal antibodies, humanized antibodies, chimeric antibodies, single-chain antibodies, Fab fragments, single-chain variable fragments (scFv), recombinant antibodies, recombinant monoclonal antibodies, aptamers, single-domain antibodies (VHHs, nanobodies), or other TMPRSS6-binding fragments or variants. In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein may comprise frameworks in which amino acids have been substituted within an existing antibody framework to affect properties such as, among other things, antigen-binding ability. In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein may comprise complementarity-determining regions (CDRs) from a source (parent) antibody grafted (fused) onto a framework of a different type (class) of antibody and / or derived from a different organism than the parent antibody, particularly an acceptor human framework. In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein may comprise frameworks in which amino acids have been substituted, mutated, or replaced in regions outside the CDRs, for example, in the variable region frameworks surrounding the CDRs and / or in the constant region, particularly the Fc region, to affect properties such as antigen binding or antibody structure. In certain embodiments, one or more CDRs have been substituted, mutated, or replaced. In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein may be humanized anti-TMPRSS6 antibody variants.

[0013] In certain embodiments, anti-TMPRSS6 antibodies disclosed herein comprise at least one polypeptide having an amino acid sequence set forth in Table 1, Table 2, or Table 3, or a sequence substantially identical (e.g., at least 85%, 90%, 92%, 95%, 97%, or 98%, 99% identical) to an amino acid sequence set forth in Table 1, Table 2, or Table 3. Anti-TMPRSS6 antibodies disclosed herein may comprise at least one polypeptide having an amino acid sequence selected from the following, or a sequence substantially identical (e.g., at least 85%, 90%, 92%, 95%, 97%, or 98%, 99% identical) to at least one polypeptide having an amino acid sequence selected from the following: SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:7; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:11; SEQ ID NO:12; SEQ ID NO:13; SEQ ID NO:14; SEQ ID NO:16; SEQ ID NO:17; SEQ ID NO:18; SEQ ID NO:19; SEQ ID NO:21; SEQ ID NO:22; SEQ ID NO:23; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:27; SEQ ID NO:28; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:32; SEQ ID NO:33; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:37; SEQ ID NO:38; SEQ ID NO:39; SEQ ID NO:41; SEQ ID NO:42; SEQ ID NO:43; SEQ ID NO:44; SEQ ID NO:46; SEQ ID NO:47; SEQ ID NO:48; SEQ ID NO:49; SEQ ID NO:51; SEQ ID NO:52; SEQ ID NO:53; SEQ ID NO:54; SEQ ID NO:56; SEQ ID NO:57; SEQ ID NO:58; SEQ ID NO:59; SEQ ID NO:61; SEQ ID NO:63; SEQ ID NO:65; SEQ ID NO:67; SEQ ID NO:69; SEQ ID NO:71; SEQ ID NO:73; SEQ ID NO:75; SEQ ID NO:77; SEQ ID NO:79; SEQ ID NO:81; or SEQ ID NO:83.

[0014] In one embodiment, the anti-TMPRSS6 antibody disclosed herein comprises a heavy chain (HC) variable region polypeptide having the amino acid sequence set forth in SEQ ID NO:1 or a sequence substantially identical to SEQ ID NO:1, and a light chain (LC) variable region polypeptide having the amino acid sequence set forth in SEQ ID NO:6 or a sequence substantially identical to SEQ ID NO:6. In one embodiment, the anti-TMPRSS6 antibody disclosed herein comprises a heavy chain complementarity determining region 1 (HC CDR1) having the amino acid sequence GYTFTSYW set forth in SEQ ID NO:2, a heavy chain complementarity determining region 2 (HC CDR2) having the amino acid sequence IYPGSGST set forth in SEQ ID NO:3, and a heavy chain complementarity determining region 3 (HC CDR3) having the amino acid sequence APYDSDYAMDY set forth in SEQ ID NO:4; a light chain complementarity determining region 1 (LC CDR1) having the amino acid sequence QDINNY set forth in SEQ ID NO:7, a light chain complementarity determining region 2 (LC CDR2) having the amino acid sequence RAN set forth in SEQ ID NO:8, and a light chain complementarity determining region 3 (LC CDR3) having the amino acid sequence LQYDEFPLT set forth in SEQ ID NO:9, or a variant of the above antibodies comprising 1, 2, 3, 4, 5, or 6 amino acid substitutions in the CDR regions. In one non-limiting embodiment, the anti-TMPRSS6 antibody disclosed herein is the antibody identified herein as MWTx-001, which comprises an HC polypeptide having the amino acid sequence set forth in SEQ ID NO: 61 and an LC polypeptide having the amino acid sequence set forth in SEQ ID NO: 63.

[0015] In one embodiment, an anti-TMPRSS6 antibody disclosed herein comprises an HC variable region polypeptide having the amino acid sequence set forth in SEQ ID NO: 11, or a sequence substantially identical to SEQ ID NO: 11, and an LC variable region polypeptide having the amino acid sequence set forth in SEQ ID NO: 16, or a sequence substantially identical to SEQ ID NO: 16. In one embodiment, an anti-TMPRSS6 antibody disclosed herein comprises an HC CDR1 having the amino acid sequence GFNIKDYY set forth in SEQ ID NO: 12, an HC CDR2 having the amino acid sequence IDPEDGES set forth in SEQ ID NO: 13, an HC CDR3 having the amino acid sequence TRGDSMMVTYFDY set forth in SEQ ID NO: 14; an LC CDR1 having the amino acid sequence QDVSTA set forth in SEQ ID NO: 17, an LC CDR2 having the amino acid sequence WAF set forth in SEQ ID NO: 18, and an LC CDR3 having the amino acid sequence QQHYRSPWT set forth in SEQ ID NO: 19, or a variant of the foregoing antibodies comprising 1, 2, 3, 4, 5, or 6 amino acid substitutions in the CDR regions. In one non-limiting embodiment, the anti-TMPRSS6 antibody disclosed herein is that of the antibody identified herein as MWTx-002, which comprises an HC polypeptide having the amino acid sequence set forth in SEQ ID NO: 65 and an LC polypeptide having the amino acid sequence set forth in SEQ ID NO: 67.

[0016] In one embodiment, an anti-TMPRSS6 antibody disclosed herein comprises an HC variable region polypeptide having the amino acid sequence set forth in SEQ ID NO: 21, or a sequence substantially identical to SEQ ID NO: 21, and an LC variable region polypeptide having the amino acid sequence set forth in SEQ ID NO: 26, or a sequence substantially identical to SEQ ID NO: 26. In one embodiment, an anti-TMPRSS6 antibody disclosed herein comprises an HC CDR1 having the amino acid sequence GFNIEDYY set forth in SEQ ID NO: 22, an HC CDR2 having the amino acid sequence IDPEDGET set forth in SEQ ID NO: 23, an HC CDR3 having the amino acid sequence ARSIYLDPMDY set forth in SEQ ID NO: 24; an LC CDR1 having the amino acid sequence QDVTTA set forth in SEQ ID NO: 27, an LC CDR2 having the amino acid sequence WAT set forth in SEQ ID NO: 28, and an LC CDR3 having the amino acid sequence QQHYSTPYT set forth in SEQ ID NO: 29, or a variant of the foregoing antibodies comprising 1, 2, 3, 4, 5, or 6 amino acid substitutions in the CDR regions. In one non-limiting embodiment, the anti-TMPRSS6 antibody disclosed herein is the antibody identified herein as MWTx-003, which comprises an HC polypeptide having the amino acid sequence set forth in SEQ ID NO: 69 and an LC polypeptide having the amino acid sequence set forth in SEQ ID NO: 71.

[0017] In one embodiment, an anti-TMPRSS6 antibody disclosed herein comprises an HC variable region polypeptide having the amino acid sequence set forth in SEQ ID NO: 31, or a sequence substantially identical to SEQ ID NO: 31, and an LC variable region polypeptide having the amino acid sequence set forth in SEQ ID NO: 36, or a sequence substantially identical to SEQ ID NO: 36. In one embodiment, an anti-TMPRSS6 antibody disclosed herein comprises an HC CDR1 having the amino acid sequence GYTFTSYW set forth in SEQ ID NO: 32, an HC CDR2 having the amino acid sequence IYPGSGST set forth in SEQ ID NO: 33, an HC CDR3 having the amino acid sequence APYDADYAMDY set forth in SEQ ID NO: 34; an LC CDR1 having the amino acid sequence QDISNY set forth in SEQ ID NO: 37, an LC CDR2 having the amino acid sequence RAN set forth in SEQ ID NO: 38, and an LC CDR3 having the amino acid sequence LQYDEFPLT set forth in SEQ ID NO: 39, or a variant of the foregoing antibodies comprising 1, 2, 3, 4, 5, or 6 amino acid substitutions in the CDR regions. In one non-limiting embodiment, the anti-TMPRSS6 antibody disclosed herein is the antibody identified herein as humanized anti-TMPRSS6 antibody variant hzMWTx-001Var, which comprises an HC polypeptide having the amino acid sequence set forth in SEQ ID NO: 73 and an LC polypeptide having the amino acid sequence set forth in SEQ ID NO: 75.

[0018] In one embodiment, an anti-TMPRSS6 antibody disclosed herein comprises an HC variable region polypeptide having the amino acid sequence set forth in SEQ ID NO: 41, or a sequence substantially identical to SEQ ID NO: 41, and an LC variable region polypeptide having the amino acid sequence set forth in SEQ ID NO: 46, or a sequence substantially identical to SEQ ID NO: 46. In one embodiment, an anti-TMPRSS6 antibody disclosed herein comprises an HC CDR1 having the amino acid sequence GFNIKDYY set forth in SEQ ID NO: 42, an HC CDR2 having the amino acid sequence IDPEDAES set forth in SEQ ID NO: 43, an HC CDR3 having the amino acid sequence TRGDSMMVTYFDY set forth in SEQ ID NO: 44; an LC CDR1 having the amino acid sequence QDVSTA set forth in SEQ ID NO: 47, an LC CDR2 having the amino acid sequence WAF set forth in SEQ ID NO: 48, and an LC CDR3 having the amino acid sequence QQHYRSPWT set forth in SEQ ID NO: 49, or a variant of the foregoing antibodies comprising 1, 2, 3, 4, 5, or 6 amino acid substitutions in the CDR regions. In one non-limiting embodiment, the anti-TMPRSS6 antibody disclosed herein is the antibody identified herein as humanized anti-TMPRSS6 antibody variant hzMWTx-002Var, which comprises an HC polypeptide having the amino acid sequence set forth in SEQ ID NO: 77 and an LC polypeptide having the amino acid sequence set forth in SEQ ID NO: 79.

[0019] In one embodiment, an anti-TMPRSS6 antibody disclosed herein comprises an HC variable region polypeptide having the amino acid sequence set forth in SEQ ID NO: 51, or a sequence substantially identical to SEQ ID NO: 51, and an LC variable region polypeptide having the amino acid sequence set forth in SEQ ID NO: 56, or a sequence substantially identical to SEQ ID NO: 56. In one embodiment, an anti-TMPRSS6 antibody disclosed herein comprises an HC CDR1 having the amino acid sequence GFNIEDYY set forth in SEQ ID NO: 52, an HC CDR2 having the amino acid sequence IDPEDAET set forth in SEQ ID NO: 53, an HC CDR3 having the amino acid sequence ARSIYLDPMDY set forth in SEQ ID NO: 54; an LC CDR1 having the amino acid sequence QDVTTA set forth in SEQ ID NO: 57, an LC CDR2 having the amino acid sequence WAT set forth in SEQ ID NO: 58, and an LC CDR3 having the amino acid sequence QQHYSTPYT set forth in SEQ ID NO: 59, or a variant of the foregoing antibodies comprising 1, 2, 3, 4, 5, or 6 amino acid substitutions in the CDR regions. In one non-limiting embodiment, the anti-TMPRSS6 antibody disclosed herein is the antibody identified herein as humanized anti-TMPRSS6 antibody variant hzMWTx-003Var, which comprises an HC polypeptide having the amino acid sequence set forth in SEQ ID NO: 81 and an LC polypeptide having the amino acid sequence set forth in SEQ ID NO: 83.

[0020] In another aspect, provided are anti-TMPRSS6 antibodies (including the variants and fragments disclosed herein) that can be used to treat disorders of iron metabolism, such as iron overload, particularly β-thalassemia and other disorders of ineffective erythropoiesis. Methods and compositions are provided for using the anti-TMPRSS6 antibodies disclosed herein for therapeutic uses, including, but not limited to, treating disorders of iron metabolism, such as iron overload, particularly β-thalassemia and other disorders of ineffective erythropoiesis. In certain embodiments, provided are pharmaceutical compositions comprising the anti-TMPRSS6 antibodies disclosed herein and a suitable carrier and / or excipient.

[0021] In another aspect, a method for treating a disorder of iron metabolism is provided, the method comprising administering an effective amount of an anti-TMPRSS6 antibody disclosed herein to a subject in need thereof, wherein the administration of the effective amount of the anti-TMPRSS6 antibody modulates the activity of a component involved in iron metabolism. In certain embodiments, a method for treating iron overload comprises administering an effective amount of an anti-TMPRSS6 antibody disclosed herein, wherein the administration of the effective amount of the anti-TMPRSS6 antibody modulates the activity of a component involved in iron metabolism. In certain embodiments, a method for treating iron overload comprises administering an effective amount of an anti-TMPRSS6 antibody disclosed herein, wherein the administration of the effective amount of the anti-TMPRSS6 antibody modulates the activity of at least one component involved in the regulation of hepcidin expression. In certain embodiments, the method comprises administering an effective amount of an anti-TMPRSS6 antibody that inhibits TMPRSS6 repression of hepcidin expression. In certain embodiments, the administration of an effective amount of the anti-TMPRSS6 antibody increases hepcidin expression. In certain embodiments, the method involves administering an effective amount of an anti-TMPRSS6 antibody that increases hepcidin promoter activity. In certain embodiments, the method involves administering an effective amount of an anti-TMPRSS6 antibody that inhibits TMPRSS6 repression of BMP / SMAD pathway-induced expression of hepcidin. In certain embodiments, the method involves administering to a subject an effective amount of an anti-TMPRSS6 antibody that produces one or more biological effects associated with iron overload, including, but not limited to, reducing serum iron, reducing liver non-heme iron, increasing serum hepcidin, increasing liver hepcidin RNA, reducing splenomegaly, increasing red blood cell count (RBC), increasing hematocrit (HCT), reducing red blood cell distribution width (RDW), and / or increasing production of mature red blood cells (increased erythropoiesis).

[0022] In another aspect, provided is a method for treating a disease or disease state associated with abnormal suppression of hepcidin expression, comprising administering an effective amount of an anti-TMPRSS6 antibody disclosed herein to a subject in need thereof, wherein the administration of the effective amount of the anti-TMPRSS6 antibody modulates the activity of at least one component involved in the abnormal suppression of hepcidin expression, thereby reducing the abnormal suppression of hepcidin expression.In certain embodiments, this method results in an increase in hepcidin expression.

[0023] In another aspect, provided is a method for treating the iron metabolism disorder associated with suppression of hepcidin level, comprising administering an effective amount of anti-TMPRSS6 antibody disclosed herein to a subject in need thereof, wherein the administration of an effective amount of anti-TMPRSS6 antibody modulates the activity of at least one component involved in the suppression of hepcidin level.In certain embodiments, the method comprises administering an effective amount of anti-TMPRSS6 antibody to increase serum hepcidin level, increase liver hepcidin RNA, and reduce serum iron level.

[0024] In another aspect, methods are provided for treating disorders of iron metabolism, including disorders associated with and / or characterized by ineffective erythropoiesis, which may include, but are not limited to, β-thalassemia. According to this aspect, such methods comprise administering an effective amount of an anti-TMPRSS6 antibody disclosed herein to a subject known to have or suspected of having a disorder of iron metabolism associated with and / or characterized by ineffective erythropoiesis, wherein the administration results in one or more changes related to iron metabolism and / or erythropoiesis in the subject. In certain embodiments, methods are provided in which administration of an effective amount of an anti-TMPRSS6 antibody treats or ameliorates at least one biological effect or symptom associated with the disorder. In certain embodiments, practicing the method results in one or more changes including, but not limited to, reducing liver non-heme iron, increasing serum hepcidin, increasing liver hepcidin RNA, reducing splenomegaly, increasing red blood cell count (RBC), increasing hematocrit (HCT), reducing red blood cell distribution width (RDW), and increasing production of mature red blood cells (increased erythropoiesis).

[0025] In another aspect, a method is provided for treating myeloproliferative disorders, including but not limited to myeloproliferative neoplasms, myeloproliferative neoplasms with a constitutively activated JAK2 / STAT5 signaling pathway, myeloproliferative disorders characterized by increased red blood cell mass and erythroid hyperplasia, polycythemia vera (PV), and / or disorders characterized by polycythemia and erythroid hyperplasia. According to this aspect, such a method comprises administering an effective amount of an anti-TMPRSS6 antibody disclosed herein to a subject known to have or suspected of having a myeloproliferative disorder. In certain embodiments, a method is provided in which administration of an effective amount of an anti-TMPRSS6 antibody treats or ameliorates at least one biological effect or symptom associated with the disorder. In certain embodiments, practicing the method results in one or more changes, including but not limited to, reduced RBCs, reduced HCT, reduced hemoglobin (HGB), reduced mean corpuscular volume (MCV), and reduced RDW, when administered to a subject known to have or suspected of having a myeloproliferative disorder. In certain embodiments, practicing the method results in one or more changes, including but not limited to, reducing RBC, reducing HCT, reducing hemoglobin (HGB), reducing mean corpuscular volume (MCV), and reducing RDW, when administered to a subject known or suspected of having polycythemia vera (PV).

[0026] In one aspect, a method for diagnosing or screening for iron overload in a subject is provided. In certain embodiments, the method includes administering an anti-TMPRSS6 antibody to a subject known to have or suspected of having iron overload, and measuring one or more biological effects or symptoms associated with iron overload.

[0027] In another aspect, a method for diagnosing or screening for a myeloproliferative disorder in a subject is provided. In certain embodiments, the method comprises administering an anti-TMPRSS6 antibody to a subject known to have or suspected of having a myeloproliferative disorder, and measuring one or more biological effects or symptoms associated with the myeloproliferative disorder.

[0028] In another aspect, one or more isolated nucleic acid molecules are provided that encode at least a portion of at least one of the anti-TMPRSS6 antibodies disclosed herein. In certain embodiments, the isolated nucleic acid molecule that encodes at least a portion of at least one of the anti-TMPRSS6 antibodies disclosed herein comprises a nucleotide sequence set forth in Table 1, Table 2, or Table 3, or a sequence that is substantially identical (e.g., at least 85%, 90%, 92%, 95%, 97%, or 98%, 99% identical) to a nucleotide sequence set forth in Table 1, Table 2, or Table 3. In certain embodiments, an isolated nucleic acid molecule encoding at least one of the heavy chain (HC) sequences of the anti-TMPRSS6 antibodies disclosed herein may comprise a nucleotide sequence selected from at least one of the following: SEQ ID NO:5 or a sequence substantially identical to SEQ ID NO:5; SEQ ID NO:15 or a sequence substantially identical to SEQ ID NO:15; SEQ ID NO:25 or a sequence substantially identical to SEQ ID NO:25; SEQ ID NO:35 or a sequence substantially identical to SEQ ID NO:35; SEQ ID NO:45 or a sequence substantially identical to SEQ ID NO:45; SEQ ID NO:55 or a sequence substantially identical to SEQ ID NO:55; SEQ ID NO:62 or a sequence substantially identical to SEQ ID NO:62; SEQ ID NO:66 or a sequence substantially identical to SEQ ID NO:66; SEQ ID NO:70 or a sequence substantially identical to SEQ ID NO:70; SEQ ID NO:74 or a sequence substantially identical to SEQ ID NO:74; SEQ ID NO:78 or a sequence substantially identical to SEQ ID NO:78; SEQ ID NO:82 or a sequence substantially identical to SEQ ID NO:82.In certain embodiments, an isolated nucleic acid molecule encoding at least one of the light chain (LC) sequences of an anti-TMPRSS6 antibody or antigen-binding fragment thereof disclosed herein may comprise a nucleotide sequence selected from at least one of the following: SEQ ID NO: 10 or a sequence substantially identical to SEQ ID NO: 10; SEQ ID NO: 20 or a sequence substantially identical to SEQ ID NO: 20; SEQ ID NO: 30 or a sequence substantially identical to SEQ ID NO: 30; SEQ ID NO: 40 or a sequence substantially identical to SEQ ID NO: 40; SEQ ID NO: 50 or a sequence substantially identical to SEQ ID NO: 50; SEQ ID NO: 60 or a sequence substantially identical to SEQ ID NO: 60; SEQ ID NO: 64 or a sequence substantially identical to SEQ ID NO: 64; SEQ ID NO: 68 or a sequence substantially identical to SEQ ID NO: 68; SEQ ID NO: 72 or a sequence substantially identical to SEQ ID NO: 72; SEQ ID NO: 76 or a sequence substantially identical to SEQ ID NO: 76; SEQ ID NO: 80 or a sequence substantially identical to SEQ ID NO: 80; SEQ ID NO: 84 or a sequence substantially identical to SEQ ID NO: 84.

[0029] In another aspect, vectors are provided that comprise one or more nucleic acid molecules encoding at least one amino acid sequence of an anti-TMPRSS6 antibody disclosed herein. In certain embodiments, vectors are provided that comprise one or more nucleic acid molecules encoding at least one of the heavy chain (HC) or light chain (LC) sequences of an anti-TMPRSS6 antibody disclosed herein. In certain embodiments, vectors are provided that comprise nucleic acid molecules that encode at least a portion of at least one of the amino acid sequences set forth in Table 1, Table 2, or Table 3, or at least a portion of an amino acid sequence substantially identical to an amino acid sequence set forth in Table 1, Table 2, or Table 3. In certain embodiments, vectors are provided that comprise nucleic acid molecules that encode at least a portion of at least one of the HC or LC sequences set forth in Table 1, Table 2, or Table 3, or at least a portion of an amino acid sequence substantially identical to at least one of the HC or LC sequences set forth in Table 1, Table 2, or Table 3.

[0030] In another aspect, at least one host cell is provided that contains a vector comprising one or more nucleic acid molecules encoding the amino acid sequence of an anti-TMPRSS6 antibody disclosed herein. In certain embodiments, a host cell is provided that contains a vector comprising a nucleic acid molecule encoding at least a portion of at least one of the HC or LC sequences listed in Table 1, Table 2, or Table 3, or at least a portion of an amino acid sequence substantially identical to at least one of the HC or LC sequences listed in Table 1, Table 2, or Table 3. In certain embodiments, at least one host cell is capable of supporting vector expression and recombinant production of an anti-TMPRSS6 antibody, or antigen-binding fragment thereof, encoded by the vector. In certain embodiments, at least one host cell is capable of supporting vector expression and recombinant production of an anti-TMPRSS6 antibody, or antigen-binding fragment thereof, encoded by a vector comprising a nucleic acid molecule encoding at least a portion of at least one of the HC or LC sequences listed in Table 1, Table 2, or Table 3, or at least a portion of an amino acid sequence substantially identical to at least one of the HC or LC sequences listed in Table 1, Table 2, or Table 3. In certain embodiments, host cells are transiently transfected with a vector comprising one or more nucleic acid molecules encoding the amino acid sequence of an anti-TMPRSS6 antibody or antigen-binding fragment thereof disclosed herein, and the host cells are capable of supporting vector expression and recombinant production of the anti-TMPRSS6 antibody or antigen-binding fragment thereof encoded by the vector.

[0031] In some aspects, the disclosure provides a method for treating polycythemia vera (PV) in a subject. In some embodiments, PV is associated with overactivation of the JAK2 / STAT5 pathway. In some embodiments, the method comprises administering to the subject an effective amount of an anti-TMPRSS6 antibody. In some embodiments, the antibody comprises a heavy chain complementarity determining region 1 (HC CDR1) comprising the amino acid sequence of SEQ ID NO: 52, a HC CDR2 comprising the amino acid sequence of SEQ ID NO: 53, a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 54, a light chain complementarity determining region 1 (LC CDR1) comprising the amino acid sequence of SEQ ID NO: 57, a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 58, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO: 59. In some embodiments, the antibody comprises a HC CDR1 comprising the amino acid sequence of SEQ ID NO: 2, a HC CDR2 comprising the amino acid sequence of SEQ ID NO: 3, a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 4, a LC CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the antibody comprises an HC CDR1 comprising the amino acid sequence of SEQ ID NO: 12, an HC CDR2 comprising the amino acid sequence of SEQ ID NO: 13, an HC CDR3 comprising the amino acid sequence of SEQ ID NO: 14, an LC CDR1 comprising the amino acid sequence of SEQ ID NO: 17, an LC CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and an LC CDR3 comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antibody comprises an HC CDR1 comprising the amino acid sequence of SEQ ID NO: 22, an HC CDR2 comprising the amino acid sequence of SEQ ID NO: 23, an HC CDR3 comprising the amino acid sequence of SEQ ID NO: 24, an LC CDR1 comprising the amino acid sequence of SEQ ID NO: 27, an LC CDR2 comprising the amino acid sequence of SEQ ID NO: 28, and an LC CDR3 comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the antibody comprises an HC CDR1 comprising the amino acid sequence of SEQ ID NO: 32, an HC CDR2 comprising the amino acid sequence of SEQ ID NO: 33, an HC CDR3 comprising the amino acid sequence of SEQ ID NO: 34, an LC CDR1 comprising the amino acid sequence of SEQ ID NO: 37, an LC CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and an LC CDR3 comprising the amino acid sequence of SEQ ID NO: 39.In some embodiments, the antibody comprises an HC CDR1 comprising the amino acid sequence of SEQ ID NO: 42, an HC CDR2 comprising the amino acid sequence of SEQ ID NO: 43, an HC CDR3 comprising the amino acid sequence of SEQ ID NO: 44, an LC CDR1 comprising the amino acid sequence of SEQ ID NO: 47, an LC CDR2 comprising the amino acid sequence of SEQ ID NO: 48, and an LC CDR3 comprising the amino acid sequence of SEQ ID NO: 49.

[0032] In some aspects, the disclosure provides a method for treating polycythemia vera (PV) in a subject. In some embodiments, the subject has bone marrow containing cells with JAK2 / STAT5 hyperactivation. In some embodiments, the method comprises administering to the subject an effective amount of an anti-TMPRSS6 antibody. In some embodiments, the antibody comprises a heavy chain complementarity determining region 1 (HC CDR1) comprising the amino acid sequence of SEQ ID NO: 52, a HC CDR2 comprising the amino acid sequence of SEQ ID NO: 53, a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 54, a light chain complementarity determining region 1 (LC CDR1) comprising the amino acid sequence of SEQ ID NO: 57, a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 58, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO: 59. In some embodiments, the antibody comprises a HC CDR1 comprising the amino acid sequence of SEQ ID NO: 2, a HC CDR2 comprising the amino acid sequence of SEQ ID NO: 3, a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 4, a LC CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the antibody comprises an HC CDR1 comprising the amino acid sequence of SEQ ID NO: 12, an HC CDR2 comprising the amino acid sequence of SEQ ID NO: 13, an HC CDR3 comprising the amino acid sequence of SEQ ID NO: 14, an LC CDR1 comprising the amino acid sequence of SEQ ID NO: 17, an LC CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and an LC CDR3 comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antibody comprises an HC CDR1 comprising the amino acid sequence of SEQ ID NO: 22, an HC CDR2 comprising the amino acid sequence of SEQ ID NO: 23, an HC CDR3 comprising the amino acid sequence of SEQ ID NO: 24, an LC CDR1 comprising the amino acid sequence of SEQ ID NO: 27, an LC CDR2 comprising the amino acid sequence of SEQ ID NO: 28, and an LC CDR3 comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the antibody comprises an HC CDR1 comprising the amino acid sequence of SEQ ID NO: 32, an HC CDR2 comprising the amino acid sequence of SEQ ID NO: 33, an HC CDR3 comprising the amino acid sequence of SEQ ID NO: 34, an LC CDR1 comprising the amino acid sequence of SEQ ID NO: 37, an LC CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and an LC CDR3 comprising the amino acid sequence of SEQ ID NO: 39.In some embodiments, the antibody comprises an HC CDR1 comprising the amino acid sequence of SEQ ID NO: 42, an HC CDR2 comprising the amino acid sequence of SEQ ID NO: 43, an HC CDR3 comprising the amino acid sequence of SEQ ID NO: 44, an LC CDR1 comprising the amino acid sequence of SEQ ID NO: 47, an LC CDR2 comprising the amino acid sequence of SEQ ID NO: 48, and an LC CDR3 comprising the amino acid sequence of SEQ ID NO: 49.

[0033] In some embodiments, the subject has a mutation that leads to overactivation of JAK2 / STAT5.

[0034] In some embodiments, the subject has a JAK2 mutation.

[0035] In some embodiments, the JAK2 mutation is a JAK2 gene exon 14 mutation. In some embodiments, the JAK2 exon 14 mutation is V617F, H606Q, H608Y, L611V, L611S, V617I, C618F, C618R, or the absence of exon 14. In some embodiments, the JAK2 exon 14 mutation is V617F. In some embodiments, the subject is a homozygote of JAK2 V617F mutation.

[0036] In some embodiments, the JAK2 mutation is a JAK2 gene exon 12 mutation. In some embodiments, the JAK2 mutation is F537-K539delinsL, N542-E543del mutation, H538QK539L, V536-I546 dup11, V536-F547 dup, F537-I546dup10F547L, F537IK539I, H538-K539delinsL, H538-K539del, H538DK539LI540S, H538G, K539L, K539E, I540-E543delinsMK, I540-E542delinsS, R541-E543delinsK, N542-E543del, D544-L545del, or 547insLI540-F547dup8.

[0037] In some embodiments, the subject has a mutation in JAK2 gene exon 15. In some embodiments, the JAK2 gene exon 15 mutation is L642P or I645V.

[0038] In some embodiments, the subject has a non-Jak2 mutation. In some embodiments, the subject has a mutation in the SRSF2 gene, SF3B1 gene, U2AF1 gene, U2AF1 gene, ZRSR2 gene, TET2 gene, DNMT3a gene, IDH1 / IDH2 gene, ASXL1 gene, EZH2 gene, LNK / SH2B3 gene, NF-E2 gene, NF1 gene, CBL gene, FLT3 gene, ERBB gene, PPM1D gene, TR53 gene, RUNX1 gene, CUX1 gene, ETV6 gene The child has a mutation in the CALR gene, MPL gene, let-7a gene, miR-26b gene, miR-27b gene, miR-28 gene, miR-30b gene, miR-30c gene, miR-125-5p gene, miR-125b-5p gene, miR-143 gene, miR-145 gene, miR-150 gene, miR-182 gene, miR-223 gene, miR-342 gene, or miR-451 gene.

[0039] In some embodiments, the mutation is an acquired mutation, a familial mutation, or a congenital mutation.

[0040] In some embodiments, the subject comprises hematopoietic progenitor cells comprising one or more mutations. In some embodiments, the one or more mutations are CD34 + CD38 - In some embodiments, one or more mutations occur in hematopoietic progenitor cells. In some embodiments, one or more mutations occur in myeloid progenitor cells. In some embodiments, one or more mutations occur in megakaryocyte-erythroid progenitor cells. In some embodiments, the subject exhibits a phenotypic profile of PV before administration.

[0041] In some embodiments, the subject has an increased hematocrit (HCT) compared to a subject without PV. In some embodiments, the subject has splenomegaly prior to administration. In some embodiments, the subject has erythrocytosis prior to administration. In some embodiments, the subject has leukocytosis prior to administration. In some embodiments, the subject has thrombocytosis prior to administration. In some embodiments, the subject has increased hemoglobin compared to a subject without PV prior to administration. In some embodiments, the subject has an increased red blood cell distribution width (RDW) compared to a subject without PV prior to administration.

[0042] In some embodiments, administering the antibody increases serum hepcidin. In some embodiments, administering the antibody reduces liver iron. In some embodiments, administering the antibody reduces HCT. In some embodiments, administering the antibody reduces red blood cell count. In some embodiments, administering the antibody reduces red blood cell distribution width (RDW). In some embodiments, administering the antibody reduces serum iron. In some embodiments, administering the antibody reduces leukocytosis. In some embodiments, administering the antibody reduces early erythroid progenitors. In some embodiments, administering the antibody reduces plasma hemoglobin levels. In some embodiments, administering the antibody reduces mean corpuscular volume (MCV). In some embodiments, administering the antibody reduces the frequency of thrombotic events (TE). In some embodiments, administering the antibody reduces the frequency of phlebotomy. In some embodiments, administering the antibody reduces the frequency of cytoreductive therapy. In some embodiments, the antibody treats a subject in need thereof who is refractory to phlebotomy and / or cytoreductive therapy. In some embodiments, administration of the antibody results in a reduction in symptoms as documented by the Myeloproliferative Neoplasm Symptom Assessment Form (MPN-SAF).

[0043] In some embodiments, the subject is administered an antibody comprising a heavy chain complementarity determining region 1 (HC CDR1) comprising the amino acid sequence of SEQ ID NO: 52, a HC CDR2 comprising the amino acid sequence of SEQ ID NO: 53, a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 54, a light chain complementarity determining region 1 (LC CDR1) comprising the amino acid sequence of SEQ ID NO: 57, a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 58, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO: 59.

[0044] In some embodiments, the subject is administered an antibody comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO:51, and a light chain variable region (VL) comprising an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO:56. In some embodiments, a subject is administered an antibody comprising a VH comprising an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO:1, and a VL comprising an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO:6.In some embodiments, the subject is administered an antibody comprising a VH comprising an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 11, and a VL comprising an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the subject is administered an antibody comprising a VH comprising an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO:21, and a VL comprising an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO:26.In some embodiments, the subject is administered an antibody comprising a VH comprising an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 31, and a VL comprising an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 36. In some embodiments, a subject is administered an antibody comprising a VH comprising an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 41, and a VL comprising an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 46. In some embodiments, the sequence diversity occurs in the framework regions of the VH and / or the framework regions of the VL of the anti-TMPRSS6 antibodies described herein.

[0045] In some embodiments, the subject is administered an antibody comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:51 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:56.

[0046] In some embodiments, a subject is administered an antibody comprising a heavy chain comprising an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 81, and a light chain comprising an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 83. In some embodiments, a subject is administered an antibody comprising a heavy chain comprising an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 61, and a light chain comprising an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 63.In some embodiments, a subject is administered an antibody comprising a heavy chain comprising an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO:65, and a light chain comprising an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO:67. In some embodiments, a subject is administered an antibody comprising a heavy chain comprising an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 69, and a light chain comprising an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 71.In some embodiments, a subject is administered an antibody comprising a heavy chain comprising an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 73, and a light chain comprising an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 75. In some embodiments, a subject is administered an antibody comprising a heavy chain comprising an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 77, and a light chain comprising an amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 79. In some embodiments, the sequence diversity occurs in the VH framework regions, VL framework regions, heavy chain constant region, and / or light chain constant region of the anti-TMPRSS6 antibodies described herein.

[0047] In some embodiments, the subject is administered an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:81 and a light chain comprising the amino acid sequence of SEQ ID NO:83.

[0048] In some embodiments, the antibody cross-reacts with at least one non-human TMPRSS6. In some embodiments, the non-human TMPRSS6 is mouse TMPRSS6 or non-human primate TMPRSS6. In some embodiments, the antibody specifically binds to human TMPRSS6. In some embodiments, the antibody does not specifically bind to human matriptase-1 or human matriptase-3.

[0049] In some embodiments, the subject receives one or more additional therapeutic agents for treating PV. In some embodiments, the additional therapeutic agents for treating PV include interferon (e.g., lopeginterferon a-2b-njft (Besremi), pegylated interferon), JAK2 inhibitors (e.g., ruxolitinib, XL019, fedratinib (SAR302503), momelotinib), JAK1 inhibitors (e.g., itacitinib), hepcidin memory drugs (e.g., rusfertide (PTG-300)), lysine-specific demethylase inhibitors (e.g., bomedemstat (IMG-7298)), TMPRSS6 antagonists (e.g., sapablursen (ISI)), and the like. 702843), SLN124], anti-TfR1 antibodies (e.g., PPMX-T003), MDM2 inhibitors [e.g., Idasanutlin (RG7388), KRT-232], tyrosine kinase inhibitors [e.g., Dasatinib, Erlotinib, Gleevec, Lestaurtinib (CEP-701)], HDAC inhibitors [e.g., Givinostat (ITF2357), MK-0683], PI3K inhibitors [e.g., Umbralisib (TGR-1202)], telomerase inhibitors (e.g., Imetelstat), phlebotomy, low-dose aspirin, or hydroxyurea. [Brief explanation of the drawings]

[0050] [Figure 1]Figure 1 shows the results of a cascade screen of anti-TMPRSS6 antibodies. Antibodies that bind to human TMPRSS6 were evaluated using an in vitro functional assay of HAMP promoter activity, and antibodies that show an effect on HAMP promoter activity were evaluated for cross-reactivity with non-human TMPRSS6. [Figure 2] Figures 2A-2F show the effect of anti-TMPRSS6 antibodies on HAMP promoter activity measured by a dual-luciferase reporter assay performed in HepG2 cells across a range of antibody concentrations. In each plot, open circles represent results using anti-TMPRSS6 antibodies, and open squares represent results using the same concentrations of mouse IgG or human IgG1 as negative (nonspecific binding) controls. Figure 2A shows the effect of the MWTx-001 anti-TMPRSS6 antibody on HAMP promoter activity across a range of antibody concentrations. Figure 2B shows the effect of the MWTx-002 anti-TMPRSS6 antibody on HAMP promoter activity across a range of antibody concentrations. Figure 2C shows the effect of the MWTx-003 anti-TMPRSS6 antibody on HAMP promoter activity across a range of antibody concentrations. Figure 2D shows the effect of the hzMWTx-001Var anti-TMPRSS6 antibody on HAMP promoter activity across a range of antibody concentrations. Figure 2E shows the effect of hzMWTx-002Var anti-TMPRSS6 antibody on HAMP promoter activity across a range of antibody concentrations, and Figure 2F shows the effect of hzMWTx-003Var anti-TMPRSS6 antibody on HAMP promoter activity across a range of antibody concentrations. [Figure 3]Figures 3A-3M show the results of determining the binding affinity of anti-TMPRSS6 antibodies. Figures 3A-3F show the results of determining the binding affinity of anti-TMPRSS6 antibodies to human TMPRSS6 expressed on HEK293T cells using two different methods. In each plot, open circles represent the results using a range of anti-TMPRSS6 antibodies, while open squares represent the results using the same concentration of mouse IgG as a negative control. Figures 3A-3C show the results of measuring the binding of MWTx-001 (Figure 3A), MWTx-002 (Figure 3B), and MWTx-003 (Figure 3C) to human TMPRSS6 using cell surface ELISA (measuring an HRP-conjugated secondary antibody) and calculating the EC50 value for each antibody as an estimate of binding affinity. Figures 3D-3F show the results of measuring the binding of MWTx-001 (Figure 3D), MWTx-002 (Figure 3E), and MWTx-003 (Figure 3F) to human TMPRSS6 using FACS (measuring an APC-conjugated secondary antibody) and calculating the EC50 value for each antibody as an estimate of binding affinity. Figures 3G-3M show the results of determining the affinity and binding kinetics of anti-TMPRSS6 antibodies to human ecto-TMPRSS6-FLAG using Octet® RED96e at analyte concentrations of 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.13 nM, 1.56 nM, and 0.78 nM. Figure 3G shows the binding kinetics of the MWTx-001 anti-TMPRSS6 antibody to ecto-TMPRSS6-FLAG. Figure 3H shows the binding kinetics of the MWTx-002 anti-TMPRSS6 antibody to ecto-TMPRSS6-FLAG. Figure 3I shows the binding kinetics of the MWTx-003 anti-TMPRSS6 antibody to ecto-TMPRSS6-FLAG. Figure 3J shows the binding kinetics of the hzMWTx-001Var anti-TMPRSS6 antibody to ecto-TMPRSS6-FLAG. Figure 3K shows the binding kinetics of the hzMWTx-002Var anti-TMPRSS6 antibody to ecto-TMPRSS6-FLAG. Figure 3L shows the binding kinetics of the hzMWTx-003Var anti-TMPRSS6 antibody to ecto-TMPRSS6-FLAG. Figure 3M summarizes the affinity measurements of all anti-TMPRSS6 antibodies. [Figure 4]Figures 4A-4U show the cross-reactivity of anti-TMPRSS6 antibodies. Figures 4A-4I show the cross-reactivity of anti-TMPRSS6 antibodies MWTx-001, MWTx-002, and MWTx-003 to human and non-human TMPRSS6 expressed on HEK293T cells. Each histogram plot shows the FACS results of a single antibody incubated with HEK293T cells expressing a TMPRSS6 target (thin line and light fill; indicated by the antibody name) and the same antibody incubated with control HEK293T cells not expressing TMPRSS6 protein (thick line and dark fill; indicated by Ctrl). Figures 4A-4C show the results of using HEK293T cells stably expressing human TMPRSS6 [HuTMPRSS6-(His)6] with MWTx-001 (Figure 4A), MWTx-002 (Figure 4B), and MWTx-003 (Figure 4C). Figures 4D-4F show the results of using HEK293T cells stably expressing mouse TMPRSS6 [MoTMPRSS6-(His)6] with MWTx-001 (Figure 4D), MWTx-002 (Figure 4E), and MWTx-003 (Figure 4F). Figures 4G-4I show the results of using HEK293T cells transiently expressing cynomolgus monkey TMPRSS6 [CynoTMPRSS6-(His)6] with MWTx-001 (Figure 4G), MWTx-002 (Figure 4H), and MWTx-003 (Figure 4I). Figures 4J-4U show that anti-TMPRSS6 antibodies cross-react with non-human [mouse (Figure 4J, Figure 4L, Figure 4N, Figure 4P, Figure 4R, Figure 4T) or cynomolgus monkey (Figure 4K, Figure 4M, Figure 4O, Figure 4Q, Figure 4S, Figure 4U)] TMPRSS6 expressed on HEK293T cells, using cell surface ELISA (measuring HRP-conjugated secondary antibody) to measure the activity of MWTx-001 anti-TMPRSS6 antibody (Figure 4J-4K), MWTx-002 anti-TMPRSS6 antibody (Figure 4J-4K), and MWTx-003 anti-TMPRSS6 antibody (Figure 4J-4K). The results of measuring the binding of the WTx-002 anti-TMPRSS6 antibody (Figures 4L-4M), MWTx-003 anti-TMPRSS6 antibody (Figures 4N-4O), hzMWTx-001Var anti-TMPRSS6 antibody (Figures 4P-4Q), hzMWTx-002Var anti-TMPRSS6 antibody (Figures 4R-4S), and hzMWTx-003Var anti-TMPRSS6 antibody (Figures 4T-4U) to non-human TMPRSS6 are shown.In each plot, open circles represent the results using anti-TMPRSS6 antibodies, and open squares represent the results using mouse IgG or human IgG1 as negative (non-specific binding) controls (the calculated EC50 of each antibody was used as an estimate of binding affinity). [Figure 5] Figures 5A–5R show the results of FACS analysis of the binding of the anti-TMPRSS6 monoclonal antibodies MWTx-001 (Figures 5A–5C), MWTx-002 (Figures 5D–5F), and MWTx-003 (Figures 5G–5I) anti-TMPRSS6 antibodies and their humanized variants hzMWTx-001Var (Figures 5J–5L), hzMWTx-002Var (Figures 5M–5O), and hzMWTx-003Var (Figures 5P–5R) anti-TMPRSS6 antibodies to HEK293T cells expressing homologous matriptase. HEK293T cells stably expressing human TMPRSS6 (matriptase-2) (Figure 5A, 5D, 5G, 5J, 5M, 5P) were used as a positive control, and HEK293T cells overexpressing matriptase (ST14) (Figure 5B, 5E, 5H, 5K, 5N, 5Q) and / or matriptase-3 (TMPRSS7) (Figure 5C, 5F, 5I, 5L, 5O, 5R) proteins were used to test binding to homologous matriptase. In each panel (Figure 5A-5R), HEK293T cells not expressing matriptase (HEK293T) were used as a negative control, and the results of the control (Ctrl) were indicated. [Figure 6]Figures 6A-6L show that anti-TMPRSS6 antibody treatment increases hepcidin expression in mice in a dose-dependent manner. Figures 6A-6C show the effects of the MWTx-003 anti-TMPRSS6 antibody (Figures 6A-6B) or its humanized variant, hzMWTx-003Var anti-TMPRSS6 antibody (Figure 6C), on serum iron. Figure 6D shows the effect of GFP-TMPRSS6 on serum hepcidin. Figures 6D-6F show the effect of the MWTx-003 anti-TMPRSS6 antibody (Figures 6D-6E) or its humanized variant, hzMWTx-003Var anti-TMPRSS6 antibody (Figure 6F), on serum hepcidin. Figure 6G shows the effect of GFP-TMPRSS6 on liver hepcidin RNA. Figures 6G-6I show the effects of the MWTx-003 anti-TMPRSS6 antibody (Figures 6G-6H) or its humanized variant, hzMWTx-003Var anti-TMPRSS6 antibody (Figure 6I), on hepatic hepcidin RNA. Figures 6J-6L show the serum concentrations of the MWTx-003 anti-TMPRSS6 antibody (Figures 6J-6K) or its humanized variant, hzMWTx-003Var anti-TMPRSS6 antibody (Figure 6L). Mouse IgG2b (MoIG2b) (Figures 6A-6B, 6D-6E, 6G-6H, 6J-6K) or human IgG1 (HuIGg1) (Figures 6C, 6F, 6I, 6L) were used as isotype controls, PBS was used as a vehicle control, and the GFP vector was used as a vector control (Figures 6A, 6D, 6G, 6J). [Figure 7]Figures 7A-7R show the in vivo efficacy of anti-TMPRSS6 antibodies using a β-thalassemia mouse model. Figures 7A-7D show the effects of MWTx-003 anti-TMPRSS6 antibodies on RBC (Figure 7A), HGB (Figure 7B), HCT (Figure 7C), and RDW (Figure 7D) using Th3 / + mice. Figure 7E shows the effect of MWTx-003 anti-TMPRSS6 antibodies on spleen weight using Th3 / + mice. Figure 7F shows the effect of MWTx-003 anti-TMPRSS6 antibodies on serum iron using Th3 / + mice. Figure 7G shows the effect of MWTx-003 anti-TMPRSS6 antibodies on liver non-heme iron using Th3 / + mice. Figure 7H shows the effect of MWTx-003 anti-TMPRSS6 antibodies on serum hepcidin using Th3 / + mice. Figure 7I shows the effect of MWTx-003 anti-TMPRSS6 antibody on liver hepcidin RNA using Th3 / + mice. Figure 7J shows the serum concentration of MWTx-003 anti-TMPRSS6 antibody using Th3 / + mice. Figures 7L-7M show the effect of MWTx-003 anti-TMPRSS6 antibody on erythropoiesis using bone marrow from Th3 / + mice. Figures 7O-7P show the effect of MWTx-003 anti-TMPRSS6 antibody on erythropoiesis using splenocytes from Th3 / + mice. Representative plots in Figures 7K-7P highlight four distinct cell clusters (I: basophilic erythroblasts, II: polychromatic erythroblasts, III: normochromatic erythroblasts and anucleated reticulocytes, and IV: mature erythrocytes) and their corresponding cell number percentages. Wild-type mice were used as positive controls (Figures 7A-7J, 7K, and 7N), and mouse IgG2b (MoIgG2b) was used as an isotype control in treatment (Figures 7A-7J, 7L, and 7O). The bar graphs in Figures 7Q-7R show the average results for cell clusters I, II, III, and IV in the bone marrow (Figure 7Q) and spleen (Figure 7R) for each treatment regime (WT, Th3 / + w / MoIgG2b, Th3 / + w / MWTx-003) after 4 weeks, where comparison identifies a shift in each population, particularly toward mature erythrocytes (cluster IV) after MWTx-003 treatment. [Figure 8]Figures 8A-8D show the results of epitope binning of the MWTx-001, MWTx-002, and MWTx-003 anti-TMPRSS6 antibodies against human ecto-TMPRSS6-FLAG using Octet® RED96e. Figure 8A shows epitope binning of the MWTx-001 anti-TMPRSS6 antibody against ecto-TMPRSS6-FLAG. Figure 8B shows epitope binning of the MWTx-002 anti-TMPRSS6 antibody against ecto-TMPRSS6-FLAG. Figure 8C shows epitope binning of the MWTx-003 anti-TMPRSS6 antibody against ecto-TMPRSS6-FLAG. Figure 8D summarizes the relative signals of the MWTx-001, MWTx-002, and MWTx-003 anti-TMPRSS6 antibodies. [Figure 9]Figures 9A-9H show the results of subchronic treatment with anti-TMPRSS6 antibodies in the Jak2V617 / + Vav-iCre mouse model of PV, where mice received IP injections of recombinant MWTx-003 (r4K12B) at dose levels of 2 mg / kg, 5 mg / kg, or 10 mg / kg, or mouse IgG2b isotype control (MoIgG2b) at 10 mg / kg every 4 days for 3 weeks and were sacrificed for analysis 4 days after the last injection; WT mice received no treatment; each symbol in the graph represents one mouse. Figures 9A-9C show the endpoint measurements of hematological parameters HCT (Figure 9A), RBC (Figure 9B), and HGB (Figure 9C) for each treatment and dose level. Figures 9D-9E also show the endpoint measurements for each treatment and dose level. Figure 9D shows splenomegaly (measured as mg / g body weight) indicating the dose-dependent development of iron-restricted erythropoiesis in mice treated with MWTx-003. Figure 9E shows serum hepcidin levels (ng / ml). Figure 9F shows serum anti-TMPRSS6 concentrations (µg / ml) at the end of the study, measured by cell surface ELISA. Figure 9G shows FACS results measuring early erythroid precursors (cluster I, basophilic erythroblasts and cluster II, polychromatic erythroblasts) in the bone marrow (top row) and spleen (bottom row) for WT (left panel, top and bottom rows), MoIgG2b isotype control (center panel, top and bottom rows), and 10 mg / kg anti-TMPRSS6 MWTx-003 treatment (right panel, top and bottom rows). Figure 9H shows Prussian blue staining images of liver (left panel) and spleen (right panel) sections from mice treated with the mouse IgG2b isotype control MoIgG2b (top row) and increasing doses of anti-TMPRSS6 MWTx-003. These images show increased iron deposition in the spleen, but no significant changes in liver iron content between treated and control mice. Obvious iron deposits are indicated by arrows. In Figures 9A-9F, ****P<0.0001, ***P<0.001, *P<0.05 using one-way ANOVA with Tukey's multiple comparison adjustment. DETAILED DESCRIPTION OF THE INVENTION

[0051] The present invention relates to novel antibodies and antigen-binding fragments thereof that bind to TMPRSS6, and methods of making and using them.

[0052] I.Terms / Definitions Scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art unless otherwise defined. Use of singular terms (such as "a," "an," "the," or other uses of singular terms) includes plural referents, and plural terms shall include the singular unless the context clearly dictates otherwise. Thus, for example, reference to an "antibody" includes "one or more" antibodies or "a plurality" of such antibodies. All publications mentioned herein are incorporated by reference in their entirety.

[0053] In general, the antibodies, antigen-binding fragments, compositions, and methods disclosed herein may employ the nomenclature and techniques of molecular biology, microbiology, cell and tissue culture, protein and nucleotide chemistry, and recombinant DNA technology available to those of skill in the art. The techniques and procedures described herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references, particularly Sambrook et al. (1989) MOLECULAR CLONING: A LABORATORY MANUAL (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) and Ausubel et al. (1994) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Volumes I-III (John Wiley & Sons, NY). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein, unless otherwise specified herein. Pharmaceutical preparations and formulations, as well as techniques and methods for the treatment of subjects, are described herein using conventional nomenclature.

[0054] "Antibody" broadly refers to a polypeptide or combination of polypeptides that recognizes and binds to an antigen via one or more immunoglobulin variable regions, which may be naturally occurring or non-naturally occurring, for example, as a result of engineering, chimerization, humanization, optimization, CDR-grafting, or affinity maturation.

[0055] The "antibody" disclosed herein may be a whole (intact, full-length) antibody, a single-chain antibody, or an antigen-binding fragment having one or two chains, and may be naturally occurring or non-naturally occurring. An antibody recognizes and binds to an antigen and includes at least sufficient complementarity-determining regions (CDRs) interspersed with framework regions (FRs). The anti-TMPRSS6 antibody disclosed herein may be, but is not limited to, at least one of a monoclonal antibody, a recombinant monoclonal antibody, a polyclonal antibody, a humanized antibody, a chimeric antibody, a single-chain antibody, a Fab fragment, a single-chain variable fragment (scFv), an aptamer, a single-domain antibody (VHH or nanobody), a recombinant antibody, a modified antibody having a peptide / other moiety and / or additional amino acids attached to the N- or C-terminus of the antibody, or other TMPRSS6-binding fragment or variant. A whole antibody, full-length antibody, intact antibody, naturally occurring antibody, or equivalent terms refer to a polypeptide, particularly a glycoprotein, comprising at least two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds. Each HC is composed of a heavy chain variable region (VH) and an HC constant region (CH), and each light chain is composed of a light chain variable region (VL) and an LC constant region (CL). The HC and LC variable regions, VH and VL, contain binding domains that interact with antigens. The VH and VL regions can be further subdivided into CDR regions, typically characterized by hypervariability, interspersed with more conserved FR regions. Each VH and VL typically consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant regions of an antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system and the classical complement system. Typically, an antibody comprises at least the sequences of heavy chain (HC) CDR1, CDR2, and CDR3 and light chain (LC) CDR1, CDR2, and CDR3, any one of which may be naturally occurring or non-naturally occurring. Fewer CDR sequences may be present, as long as the antibody is capable of recognizing and binding to an antigen.

[0056] The anti-TMPRSS6 antibodies disclosed herein may be variants containing at least one altered CDR or framework sequence, and the CDR and / or framework sequence may be optimized by mutating the nucleic acid molecule encoding such framework sequence. Variants may be constructed with HC and LC portions independently derived from different sources. Techniques for creating variants include, but are not limited to, conservative amino acid substitution, computer modeling, screening of candidate polypeptides alone or in combination, and codon optimization, and it is understood that those skilled in the art can create antibody variants as needed. The anti-TMPRSS6 antibodies disclosed herein may also be fragments. The antigen-binding function of an antibody can be performed by fragments such as: Fab fragments; monovalent fragments consisting of the VL, VH, CL, and CH1 domains; F(ab)2 fragments; bivalent fragments containing two Fab fragments linked by a disulfide bridge at the hinge region; Fd fragments consisting of the VH and CH1 domains; single-chain variable fragments (scFv) consisting of the VL and VH domains of a single antibody arm; single-domain antibody (dAb) fragments consisting of the VH domain; and isolated CDRs (VHH, nanobodies), or aptamers. Antigen-binding moieties can be incorporated into single-domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, for example, Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136). The antigen-binding portion of an antibody can be grafted onto a polypeptide-based scaffold to form a monobody (see, for example, US Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies).

[0057] The term antibody encompasses a wide variety of biochemically distinguishable polypeptide classes. The "class" of an antibody refers to the type of constant domain or region possessed by its heavy chain. Those skilled in the art understand that there are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, several of which can be further subdivided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, each of which is well characterized and known to be functionally specialized. Modified versions of each of these classes and isotypes are readily identifiable and are within the scope of this disclosure. While all immunoglobulin classes are within the scope of this disclosure, this disclosure is primarily directed to the IgG class of immunoglobulin molecules.

[0058] The term "chimeric" antibody refers to an antibody in which the portions of the heavy chain (HC) and / or light chain (LC) involved in forming the immune reactive site are derived from a particular source or species, while the remaining portions of the HC and / or LC are derived from a different source or species. In certain embodiments, the target binding region or site is derived from a non-human source (e.g., mouse or non-human primate) and the constant region is human.

[0059] As used herein, the term "humanized antibody" refers to an antibody or antibody variant derived from a non-human antibody, typically a mouse monoclonal antibody, in which the CDRs derived from the parent non-human antibody are grafted (fused) into a human immunoglobulin framework, particularly a framework comprising a variable region derived from an acceptor human framework or a human consensus framework. The techniques and principles for designing, producing, and testing humanized antibodies are known (Jones PT, Dear PH, Foote J, Neuberger MS, Winter G. Replacing the complementarity-determining regions in a human antibody with those from a mouse. Nature. 1986 May 29-Jun 4;321(6069):522-5; Almagro JC, Fransson J. Humanization of antibodies. Front Biosci. 2008 Jan 1;13:1619-33). It is understood that modifications can be made to the acceptor framework at multiple locations to develop humanized antibodies with improved characteristics depending on the desired application, such as, for example, higher affinity for the target, lower clearance, lower toxicity, etc. The anti-TMPRSS6 antibodies disclosed herein may be humanized variants.

[0060] "Affinity" refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, binding affinity refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). Affinity can be measured by common methods known in the art, including those described herein. The calculated concentration at approximately 50% of maximal binding (calculated EC 50 ) can be used as an estimate of affinity. The affinity of molecule X for partner Y is generally measured using the dissociation constant (Kd or KD, k off / kon (which represents the

[0061] A "subject" is a mammal, including, but not limited to, primates (e.g., humans, non-human primates such as monkeys), livestock animals (e.g., cows, sheep, cats, dogs, pigs, llamas, and horses), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the subject is a human. The phrases "to a subject in need thereof" or "to a patient in need thereof" or "to a patient in need of treatment" or "subject in need of treatment" may include subjects who would benefit from administration of an anti-TMPRSS6 antibody disclosed herein for the treatment of iron overload. Administration of an anti-TMPRSS6 antibody encompasses administration to a "subject in need thereof," and it is understood that a "subject in need thereof" may be interpreted as referring to a subject who is known to have or suspected of having iron overload, particularly β-thalassemia, based on indicators such as symptoms, family history, or genotype. It is further understood that anti-TMPRSS6 antibodies may be administered to subjects not known to or suspected of having a disorder of iron metabolism for purposes including, but not limited to, preventative or prophylactic purposes, screening purposes, diagnostic purposes, research purposes, or to obtain a result other than treating a disorder.

[0062] For example, an "effective amount" of an anti-TMPRSS6 antibody in a pharmaceutical formulation refers to an amount effective at a dosage and for a period of time necessary to achieve a desired therapeutic or prophylactic result. It is understood that "effective amount" is intended to refer to the amount of an anti-TMPRSS6 antibody or pharmaceutical composition containing an anti-TMPRSS6 antibody that induces a measured biological response in a cell, tissue, system, non-human animal subject, non-human mammalian subject, or human subject, or a desired therapeutic effect. The terms "therapeutically effective amount," "pharmacologically effective amount," and "physiologically effective amount" are used interchangeably to refer to the amount of an anti-TMPRSS6 antibody required to provide a threshold level of active agent in the bloodstream or target tissue. The exact amount depends on numerous factors, such as the specific anti-TMPRSS6 antibody (active agent), the components and physical properties of the composition, the intended population of subjects / patients to be treated, the subject's disease state, age, sex, and weight, and other considerations, and can be readily determined by one of ordinary skill in the art based on the information provided herein or otherwise available in the relevant literature. The terms "improve," "increase," or "reduce," as used herein, refer to a value or parameter relative to a baseline measurement, e.g., a measurement in the same subject prior to the initiation of a treatment described herein, or a measurement in a control individual (or control individuals) in the absence of a treatment described herein.

[0063] The terms "pharmaceutical composition" or "pharmaceutical formulation" refer to a preparation in a form that enables the biological activity of the active ingredient contained therein, particularly an anti-TMPRSS6 antibody. It is understood that a pharmaceutical composition may contain two or more active ingredients, for example, two or more anti-TMPRSS6 antibodies, or a combination of an anti-TMPRSS6 antibody and another active ingredient acting on a different target. Such combinations may include, but are not limited to, a combination of an anti-TMPRSS6 antibody and another active ingredient that has a desired effect on the hematopoietic process, particularly erythropoiesis, a combination of an anti-TMPRSS6 antibody and a gene therapy agent, such as an agent for gene therapy targeting the HBB gene, or a combination of an anti-TMPRSS6 antibody and an Fc fusion protein targeting a TGF superfamily ligand that stimulates erythropoiesis. A "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical formulation other than the active ingredient that is non-toxic to a subject. It is understood that a pharmaceutically acceptable carrier may be, but is not limited to, a buffer, excipient, stabilizer, adjuvant, or preservative.

[0064] The term "Janus kinase 2 / signal transducer and activator of transcription 5 (JAK2 / STAT5) pathway" or "JAK2 / STAT5 signaling" as used herein refers to a cell signaling pathway (e.g., involved in processes such as immunity, cell division, cell death, and tumorigenesis) mediated by Janus kinase 2 (JAK2), signal transducer and activator of transcription 5 (STAT5), JAK2-associated receptors, and other regulatory proteins. JAK2 is a member of the Janus kinase family, a non-receptor protein tyrosine kinase. JAKs (e.g., JAK2) associate with the cytoplasmic domains of several cytokine receptors (i.e., receptors lacking intrinsic kinase activity, including, but not limited to, interferon receptors, GM-CSF receptor family receptors (e.g., IL-3R, IL-5R, and GM-CSF-R), gp130 receptor family receptors (e.g., IL-6R), and single-chain receptors (e.g., Epo-R, Tpo-R, GH-R, PRL-R)). Upon cytokine binding, JAK2 is activated through tyrosine phosphorylation of the cytoplasmic domain of cytokine receptors. Activation of JAK2 promotes the recruitment of transcription factors, such as signal transducer and activator of transcription (STAT5), to the receptor complex. (See, e.g., Levine et al., Role of JAK2 in the pathogenesis and therapy of myeloproliferative disorders. Nat Rev Cancer. 2007;7:673-683.) JAK2 further phosphorylates STATs (e.g., STAT5), and JAK2-mediated STAT phosphorylation leads to the formation of stable STAT homodimers or heterodimers (e.g., STAT5 homodimers). The phosphorylated STAT dimers (e.g., STAT5 homodimers) translocate to the nucleus, bind to the promoters of downstream genes, and initiate transcription of these genes.

[0065] In some embodiments, JAKs have a pseudokinase domain (i.e., a JH2 domain) upstream of a C-terminal tyrosine kinase domain (i.e., a JH1 domain). In some embodiments, the pseudokinase domain of JAKs is important for maintaining a low basal level of tyrosine kinase activity (e.g., in the absence of cytokines) (see, e.g., Hubbard, Mechanistic Insights into Regulation of JAK2 Tyrosine Kinase, Frontiers in Endocrinology, January 2018, Volume 8, Article 361).

[0066] The term "overactivation of the JAK2 / STAT5 pathway" or "overactivation of JAK2 / STAT5," as used herein, refers to abnormal activation (e.g., increased activation, constitutive activation) of the JAK2 / STAT5 pathway compared to physiologically normal activation levels. In some embodiments, overactivation of JAK2 / STAT5 can be determined by comparing JAK2 / STAT5 activity, measured by any suitable method, with JAK2 / STAT5 activity in a normal subject. For example, in some embodiments, overactivation of JAK2 / STAT5 can be determined by comparing JAK2 / STAT5 activity in cells of a subject with a mutant JAK2 allele with JAK2 / STAT5 activity in a normal subject with only a wild-type JAK2 allele, to determine whether a JAK2 mutation is associated with overactivation. In some embodiments, similar analyses can be performed to determine the effects of other genetic alterations (e.g., in STAT5 or other genes) on JAK2 / STAT5 signaling. The activity of the JAK2 / STAT5 pathway can be measured by any suitable method known in the art, for example, kinase activity assays (e.g., JAK2 Assay Kit Catalog No. 79520 by BPS Bioscience; PathHunter® eXpress EpoR-JAK2 Functional Assay Catalog No. 93-0900E3CP by Eurofins; HTScan® Jak2 Kinase Assay Kit Catalog No. 7752 by Cell Signaling Technology, etc.); Western blot (e.g., Western blot of phosphorylated JAK2, STAT5, and / or downstream proteins); RT-PCR (e.g., RT-PCR of expression levels of downstream genes).

[0067] In some embodiments, the subject is a subject with polycythemia vera (PV). In some embodiments, PV is associated with hyperactivation of JAK2 / STAT5 hyperactivation. In some embodiments, a subject with PV has JAK2 / STAT5 activity that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% higher than the JAK2 / STAT5 activity of a subject without PV. In some embodiments, the subject is a subject with polycythemia vera (PV). In some embodiments, PV is associated with hyperactivation of JAK2 / STAT5 hyperactivation. In some embodiments, a subject with PV has JAK2 / STAT5 activity that is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, at least 75-fold, or at least 100-fold higher than the JAK2 / STAT5 activity of a subject without PV. In some embodiments, a subject with PV has JAK2 / STAT5 activity that is up to 1.5-fold, up to 2-fold, up to 3-fold, up to 4-fold, up to 5-fold, up to 6-fold, up to 7-fold, up to 8-fold, up to 9-fold, up to 10-fold, up to 15-fold, up to 20-fold, up to 25-fold, up to 50-fold, up to 75-fold, or up to 100-fold higher than the JAK2 / STAT5 activity of a subject without PV.In some embodiments, a subject with PV has JAK2 / STAT5 activity that is 10% to 100 fold, 20% to 100 fold, 30% to 100 fold, 40% to 100 fold, 50% to 100 fold, 60% to 100 fold, 70% to 100 fold, 80% to 100 fold, 90% to 100 fold, 1 to 100 fold, 5 to 100 fold, 10 to 100 fold, 20 to 100 fold, 25 to 100 fold, 50 to 100 fold, 75 to 100 fold, 10% to 90 fold, 20% to 90 fold, 30% to 90 fold, 40% to 90 fold, 50% to 90 fold, 60% to 90 fold, 70% to 90 fold, 80%~90x, 90%~90x, 1~90x, 5~90x, 10~90x, 20~90x, 25~90x, 50~90x, 75~ 90x, 10%~80x, 20%~80x, 30%~80x, 40%~80x, 50%~80x, 60%~80x, 70%~80x , 80%~80x, 90%~80x, 1~80x, 5~80x, 10~80x, 20~80x, 25~80x, 50~80x, 75 ~80x, 10%~50x, 20%~50x, 30%~50x, 40%~50x, 50%~50x, 60%~50x, 70%~50 times, 80%~50x, 90%~50x, 1~50x, 5~50x, 10~50x, 20~50x, 25~50x, 10%~25x, 20%~25x, 30%~25x, 40%~25x, 50%~25x, 60%~25x, 70%~25x, 80%~25x, 90% ~25x, 1~25x, 5~25x, 10~25x, 20~25x, 2~25x, 12~25x, 18~25x, 10%~10x, 20%~10x, 30%~10x, 40%~10x, 50%~10x, 60%~10x, 70%~10x, 80%~10x, 90% have ~10 fold, 1-10 fold, 5-10 fold, 2-10 fold, 3-10 fold, 4-10 fold, 5-10 fold, 6-10 fold, 7-10 fold, 8-10 fold, 9-10 fold, 10%-5 fold, 20%-5 fold, 30%-5 fold, 40%-5 fold, 50%-5 fold, 60%-5 fold, 70%-5 fold, 80%-5 fold, 90%-5 fold, 1-5 fold, 2-5 fold, 3-5 fold, 4-5 fold, 10%-2 fold, 20%-2 fold, 30%-2 fold, 40%-2 fold, 50%-2 fold, 60%-2 fold, 70%-2 fold, 80%-2 fold, 90%-2 fold, or 1-2 fold higher JAK2 / STAT5 activity.

[0068] As used herein, the terms "treat" or "treating" or similar terms may refer to an outcome deemed beneficial to a particular subject under a defined set of circumstances. Treating a disorder of iron metabolism may refer non-exclusively to any of reducing, ameliorating, slowing, interrupting, preventing, mitigating, halting, or reversing the progression or severity of an existing symptom, disorder, condition, or disease, and may further include preventing or delaying the onset of one or more symptoms of iron overload and / or reducing the severity or frequency of one or more symptoms of iron overload. The terms "treating" or "method of treating" or equivalents may encompass one or more uses of the anti-TMPRSS6 antibodies disclosed herein, including, but not limited to, therapeutic, prophylactic, preventative, diagnostic, imaging, and screening uses.

[0069] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating a nucleic acid to which it is linked in a host cell into which the vector is introduced. Vectors capable of directing the expression of an operably linked nucleic acid are referred to herein as "expression vectors."

[0070] II. Anti-TMPRSS6 antibody Provided are antibodies and antigen-binding fragments that can bind to TMPRSS6 on the surface of cells and modulate the activity of at least one component involved in iron metabolism, particularly at least one component involved in iron overload associated with abnormal suppression of hepcidin expression.Anti-TMPRSS6 antibodies that can bind to TMPRSS6 on the surface of cells and modulate the activity of at least one component involved in the regulation of hepcidin expression can be used in the method for treating iron overload associated with abnormal suppression of hepcidin expression.Anti-TMPRSS6 antibodies that can bind to TMPRSS6 on the surface of cells and modulate the TMPRSS6 suppression of hepcidin expression can be used to therapeutically target TMPRSS6 in the method for treating iron overload and / or other iron regulation disorders and / or iron overload associated with abnormal suppression of hepcidin expression.

[0071] Once an antibody or fragment specific to cell surface-expressed TMPRSS6, particularly human TMPRSS6, is obtained, its desired biological activity of modulating the activity of at least one component involved in iron metabolism can be tested by several methods known to those skilled in the art.

[0072] It is understood that "modulate" or "modulating" or similar terms can refer to one or more effects that may occur when an anti-TMPRSS6 antibody disclosed herein binds to its target. "Modulating" and its equivalents refer to various modes of action and effects depending on the component under consideration; i.e., modulating can refer to neutralizing, reversing, inhibiting, blocking, reducing, antagonizing, or otherwise interfering with the activity of certain components involved in iron metabolism, while for other components involved in iron metabolism, the term modulating can refer to increasing, enhancing, or having an agonistic effect on these components.

[0073] It is understood that the term "component" can refer not only to the target molecule TMPRSS6, but also to downstream processes or pathways involved in iron metabolism.Thus, the component in the sense of process or pathway can be, but is not limited to, one or more processes involved in regulating hepcidin expression, TMPRSS6 suppression of hepcidin expression, the process of hepcidin expression, regulating hepcidin level, increasing hepcidin level, hepcidin promoter activity, or TMPRSS6 suppression of hepcidin expression induced by BMP / SMAD pathway, regulating liver non-heme iron level, splenomegaly, or one or more hematopoietic processes involved in regulating red blood cell count (RBC), hematocrit (HCT), red blood cell distribution width (RDW), and erythropoiesis, particularly the production of mature red blood cells.

[0074] The anti-TMPRSS6 antibodies disclosed herein can be used to therapeutically target at least one component involved in iron metabolism, particularly at least one component involved in iron overload. In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein can be used to therapeutically target at least one component involved in regulating hepcidin expression and modulate the activity of the component to achieve increased hepcidin expression. In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein can be used to modulate the activity of the hepcidin promoter to achieve increased hepcidin expression. It is understood that the anti-TMPRSS6 antibodies disclosed herein can be used to therapeutically target TMPRSS6, thereby modulating the downstream activity of other components of hepcidin expression, including, but not limited to, regulating liver non-heme iron levels, one or more processes involved in splenomegaly, or one or more hematopoietic processes involved in regulating red blood cell count (RBC), hematocrit (HCT), red blood cell distribution width (RDW), and erythropoiesis, particularly the production of mature red blood cells.

[0075] The anti-TMPRSS6 antibody disclosed herein is used to therapeutically target at least one component involved in iron metabolism, allowing for precise modulation of the target component.It is understood that the anti-TMPRSS6 antibody disclosed herein is used to precisely target TMPRSS6 and its downstream effects on at least one component involved in the regulation of hepcidin expression, thereby avoiding the undesirable effects, delivery and / or efficacy problems, and regulatory hurdles associated with other approaches to treat iron overload that are currently in use or under development, such as blood transfusions that may further worsen iron overload, iron chelation therapy that patients do not comply with, invasive phlebotomy or splenectomy that only manage symptoms, gene therapy targeting the HBB gene that may have persistent pleiotropic effects in multiple systems, gene therapy and gene editing that have unknown off-target effects, Fc fusion proteins targeting TGF superfamily ligands to inhibit SMAD signaling that do not reduce the need for iron chelation therapy to manage iron overload, hepcidin mimetics, and antisense or iRNA drugs that target TMPRSS6, which are difficult to control or deliver. It is understood that the use of anti-TMPRSS6 antibodies for precise therapeutic targeting does not preclude the possibility of using anti-TMPRSS6 antibodies in methods and compositions for combination treatment, for example, in combination with another active ingredient that acts on a different target, in combination with an antibody that binds to a different target, in combination with gene therapy agents and methods that target the HBB gene, or in combination with an Fc fusion protein that targets a TGF superfamily ligand that stimulates erythropoiesis.

[0076] The anti-TMPRSS6 antibodies disclosed herein allow for the development of treatments that can be tailored to each subject (e.g., dosage, frequency of administration), can be easily continued and discontinued, and can be combined with other therapies. In certain strategic embodiments, the anti-TMPRSS6 antibodies disclosed herein can be used in combination with other therapies that may address multiple therapeutic targets and / or address deficiencies or undesirable effects of one of the therapies in a combined therapy.

[0077] Exemplary Embodiments of Anti-TMPRSS6 Antibodies and Uses Thereof Non-limiting exemplary embodiments of the anti-TMPRSS6 antibodies of the present invention are disclosed, inter alia, in the Examples, Tables, and Figures.

[0078] a.An antibody capable of binding to TMPRSS6 As demonstrated in the Examples, a functional cascade can be used to identify and characterize anti-TMPRSS6 antibodies of the present invention, where the first step of the cascade involves screening for antibodies capable of binding to human TMPRSS6 on the surface of TMPRSS6-expressing cells (Example 1, Figure 1), followed by a second step in which antibodies capable of binding to human TMPRSS6 on the surface of TMPRSS6-expressing cells and modulating the activity of a component involved in iron metabolism are identified and tested for their ability to increase hepcidin (HAMP) promoter activity (Example 2). As demonstrated in the exemplary embodiment shown in Figure 1, the first step identified 143 antibodies (clones) capable of binding to human TMPRSS6 on the surface of TMPRSS6-expressing cells, and the second step identified 10 antibodies (out of the 143 screened) as "active" antibodies (clones) capable of increasing hepcidin (HAMP) promoter activity.

[0079] In the third step of the functional cascade (Figure 1), the 10 "active" antibodies were tested for cross-reactivity with non-human TMPRSS6 targets from sources relevant for further study: mouse TMPRSS6, relevant for preclinical efficacy testing in mouse models, and cynomolgus monkey TMPRSS6, relevant for toxicity (safety) testing. As demonstrated in the exemplary embodiment shown in Figure 1, demonstrated in Example 4, and illustrated in Figure 4, three clones (out of the 10 screened) showed cross-reactivity with at least one non-human TMPRSS6 target and were designated MWTx-001, MWTx-002, and MWTx-003. Each monoclonal antibody was sequenced, and the CDRs on each HC and LC were identified (Kabat numbering). The HC and LC sequences were identified as follows: MWTx-001 [SEQ ID NOs: 61 (HC) and 63 (LC)]; MWTx-002 [SEQ ID NOs: 65 (HC) and 67 (LC)]; and MWTx-003 [SEQ ID NOs: 69 (HC) and 71 (LC)]. While the monoclonal antibody is isolated and sequenced from a hybridoma cell line producing the monoclonal antibody, it is understood that the antibody may be a monoclonal antibody isolated from an antibody-producing cell line or a recombinant monoclonal antibody produced by recombinant expression of the antibody's known HC and LC. The hybridoma cell line producing the MWTx-001 monoclonal antibody was deposited under the Budapest Treaty with the American Type Culture Collection (ATCC®), 10801 University Boulevard, Manassas, Virginia, 20110, United States of America, on May 27, 2020, under ATCC Accession Number PTA-126759.The hybridoma cell line producing the MWTx-002 monoclonal antibody was deposited under the Budapest Treaty with the American Type Culture Collection (ATCC®), 10801 University Boulevard, Manassas, Virginia, 20110, United States of America, on May 27, 2020, under ATCC accession number PTA-126760. The hybridoma cell line producing the MWTx-003 monoclonal antibody was deposited under the Budapest Treaty with the American Type Culture Collection (ATCC®), 10801 University Boulevard, Manassas, Virginia, 20110, United States of America, on May 27, 2020, under ATCC accession number PTA-126761.

[0080] b. Humanized variants Humanized antibodies, in which CDRs from non-human sources are grafted onto human-derived antibody frameworks, are expected to be non-immunogenic when administered to human subjects. As demonstrated by the exemplary embodiment disclosed in Example 2, humanized anti-TMPRSS6 antibody variants were successfully generated, tested, optimized, and selected. Multiple candidate HC and LC variants were developed, each with the same CDR sequences, but in which the variable region framework sequences can differ at more than 90% of the framework positions. These variants were tested in various HC / LC combinations to identify combinations with desired characteristics. After initial design and testing, variants that exhibited the desired antigen-binding affinity were selected for further evaluation and development, including, but not limited to, modifications of some parent CDR sequences to avoid potential undesirable events such as aspartic acid isomerization, and modifications of some constant region (Fc) sequences to achieve desired functions such as minimizing antibody-dependent cellular cytotoxicity (ADCC), resulting in the humanized variants hzMWTx-001Var [SEQ ID NOs: 73 (HC) and 75 (LC)], hzMWTx-002Var [SEQ ID NOs: 77 (HC) and 79 (LC)], and hzMWTx-003Var [SEQ ID NOs: 81 (HC) and 83 (LC)].

[0081] c. Anti-TMPRSS6 antibody that increases hepcidin promoter activity As disclosed herein, antibodies for use in treating iron overload characterized by decreased hepcidin expression can modulate the activity of at least one component involved in hepcidin expression, which may be the activity of the hepcidin promoter. As demonstrated by exemplary embodiments using the in vitro assay disclosed in Example 2, the anti-TMPRSS6 antibodies MWTx-001, MWTx-002, MWTx-003, hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var increased HAMP promoter activity in a dose-dependent manner (FIGS. 2A-2F), whereas the isotype control at the same concentration did not increase HAMP promoter activity.

[0082] d. Anti-TMPRSS6 antibodies with high affinity for their targets in a relevant biological context The anti-TMPRSS6 antibodies exhibited high affinity for the biologically relevant target, i.e., human TMPRSS6 expressed on the cell surface. As demonstrated by exemplary embodiments of affinity measurements using three different methods disclosed in Example 3 and Figure 3M, the monoclonal antibodies MWTx-001, MWTx-002, and MWTx-003, and the humanized variants hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var consistently exhibited favorable affinity profiles for therapeutically effective antibodies or antibody fragments.

[0083] e. Anti-TMPRSS6 antibodies with cross-reactivity with non-human targets So that the antibody or antibody fragment should recognize, for example, the mouse homolog and / or the primate homolog, such as from cynomolgus monkeys, it is desirable for the therapeutically useful antibody or antibody fragment to have sufficient cross-reactivity with a non-human target (non-human homolog) from a source relevant for further study, such as preclinical efficacy studies, animal models of disease, toxicity studies, etc. As demonstrated by the exemplary embodiment disclosed in Example 4, MWTx-001, hzMWTx-001Var, MWTx-003, and hzMWTx-003Var showed detectable cross-reactivity with mouse TMPRSS6, while MWTx-001, MWTx-002, MWTx-003, hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var showed detectable cross-reactivity with cynomolgus monkey TMPRSS6.

[0084] f. Anti-TMPRSS6 antibody specifically binds to TMPRSS6 (matriptase-2) Antibodies with high levels of specific binding to a target protein and low cross-reactivity with homologous proteins within the same organism are expected to have few or no off-target effects. The anti-TMPRSS6 antibodies provided herein exhibit high specificity for human TMPRSS6 (matriptase-2), making them suitable for use in targeting compositions and methods. As demonstrated by the exemplary embodiments disclosed in Example 5 and illustrated in Figures 5A-R, monoclonal antibodies MWTx-001, MWTx-002, MWTx-003, and their humanized variants hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var exhibited specific binding to human TMPRSS6 (matriptase-2) and no detectable cross-reactivity with homologous human matriptase; i.e., these antibodies did not exhibit detectable binding to matriptase-1 (ST14) or matriptase-3 (TMPRSS7).

[0085] g. Anti-TMPRSS6 antibodies with dose-dependent effects in vivo on hormones and symptoms associated with iron overload Antibodies capable of increasing serum hepcidin levels, a hormone that controls iron absorption and mobilization from iron stores, are expected to reduce, ameliorate, or prevent symptoms of iron overload, particularly elevated serum iron levels. As demonstrated by the exemplary embodiment shown in Example 6, administration of the anti-TMPRSS6 monoclonal antibody MWTx-003 or the humanized variant hzMWTx-003Var to wild-type subjects, i.e., subjects not known or suspected of having iron overload, resulted in increased serum hepcidin levels (Figures 6A-6C), decreased serum iron levels (Figures 6D-6F), and increased liver hepcidin RNA levels (Figures 6G-6I) compared with isotype controls. These effects were dose-dependent, which may be interpreted as indicating that, without wishing to be bound by the mechanism of action, the dose-dependent in vivo effects of anti-TMPRSS6 antibodies indicate that one skilled in the art can determine an effective amount (dosage) for a given subject.

[0086] h. Anti-TMPRSS6 antibodies for treating β-thalassemia Antibodies and antibody fragments that can alleviate one or more symptoms of iron overload in vivo when administered to subjects representing animal models of the disease, i.e., subjects known or suspected to have iron overload, are expected to be therapeutically effective in clinical use. As demonstrated by the exemplary embodiment shown in Example 7 using a Th3 / + mouse model of β-thalassemia, administration of the anti-TMPRSS6 monoclonal antibody MWTx-003 resulted in multiple effects, including, but not limited to, reduced hepatic non-heme iron, increased serum hepcidin, increased liver hepcidin RNA, reduced splenomegaly, increased red blood cell count (RBC), increased hematocrit (HCT), reduced red blood cell distribution width (RDW), and increased production of mature red blood cells (increased erythropoiesis), compared to isotype control. Each of these effects can be understood as an improvement in the symptoms of the disorder. Symptoms of this disorder manifest in multiple biological systems, including, but not limited to, effects on the liver (effects on liver non-heme iron, liver hepcidin RNA), blood (effects on serum iron levels, circulating hormone levels, particularly serum hepcidin levels, RBC, HCT, RDW), spleen size and function (splenomegaly), and erythropoiesis at multiple sites, including but not limited to the bone marrow and spleen (effects on the abundance of different progenitor cell types and the abundance of mature red blood cells at the site of erythropoiesis). Administration of anti-TMPRSS6 antibodies improved multiple symptoms across disease model subjects, and measured symptom levels shifted from those seen in disease model (untreated disease) isotype controls to those seen in wild-type littermates, which represent normal levels in genetically similar subjects not known or suspected to have the disease. Without wishing to be bound by theory or mechanism of action, it is understood that ineffective erythropoiesis is the driving force behind increased iron absorption and abnormal suppression of hepcidin, leading to iron overload; therefore, treatments that improve erythroblast differentiation and maturation into red blood cells should be therapeutically beneficial in treating iron overload.Non-limiting exemplary embodiments of the present invention disclose anti-TMPRSS6 antibody therapy that increases erythroblast differentiation and maturation into red blood cells and also reduces iron load.

[0087] i. Anti-TMPRSS6 antibodies for treating polycythemia vera (PV)

[0088] Accordingly, certain embodiments of the present disclosure provide methods and compositions related thereto for treating conditions associated with abnormal iron metabolism in subjects with myeloproliferative neoplasms (MPNs) associated with JAK2 / STAT5 hyperactivation (e.g., polycythemia vera). Aspects of the present disclosure have been directed to methods and compositions (e.g., anti-TMPRSS6 antibodies) useful for regulating iron metabolism to treat polycythemia vera in subjects with JAK2 / STAT5 hyperactivation.

[0089] Polycythemia vera (PV) is a chronic myeloproliferative neoplasm. PV is characterized by erythrocytosis, bone marrow erythroid and megakaryocytic hyperplasia, fatigue, aquagenic pruritus, microvascular symptoms, and symptomatic splenomegaly. In some embodiments, excessive activation of the JAK2 / STAT5 pathway leads to unregulated proliferation of cells (e.g., hematopoietic progenitor cells), thus resulting in erythrocytosis, leukocytosis, and thrombocytosis in subjects with PV. Complications of PV include an increased risk of arterial and venous thrombosis and possible progression to myelofibrosis (MF) and MPN-blast phases (see, e.g., Polycythemia vera: the natural history of 1213 patients followed for 20 years. Gruppo Italiano Studio Policitemia. Ann Intern Med. 1995;123:656-64; Passamonti et al., Life expectancy and prognostic factors for survival in patients with polycythemia vera and essential thrombocythemia. Am J Med. 2004;117:755-61; Stein et al., Polycythemia vera: an appraisal of the biology and management 10 years after the discovery of JAK2 V617F. J Clin Oncol. 2015;33:3953-60).

[0090] In some embodiments, a subject with PV exhibits a phenotypic profile of PV before treatment (e.g., a treatment described herein). In some embodiments, the subject has erythrocytosis compared to a subject without PV. Erythrocytosis refers to excessive production of red blood cells. Erythrocytosis can be assessed by measuring hematocrit (HCT), hemoglobin, and bone marrow cell morphology in a subject (see, e.g., Mithoowani et al., Investigation and management of erythrocytosis, CMAJ. 2020 Aug 10; 192(32): E913-E918).

[0091] In some embodiments, subjects with erythrocytosis associated with PV have an increased hematocrit (HCT) compared to subjects without PV. Hematocrit is the volume percentage of red blood cells in the blood. The normal range for HCT is about 38%-48% in men and about 35%-45% in women. In some embodiments, subjects with PV have increased erythrocytosis and exhibit increased HCT levels greater than 48% in women and greater than 52% in men. In some embodiments, subjects with PV have an HCT level of more than 48%, more than 49%, more than 50%, more than 51%, more than 52%, more than 53%, more than 54%, more than 55%, more than 56%, more than 57%, more than 58%, more than 59%, more than 60%, more than 61%, more than 62%, more than 63%, more than 64%, more than 65%, more than 66%, more than 67%, more than 68%, more than 69%, more than 70%, more than 71%, more than 72%, more than 73%, more than 74%, more than 75%, or more than that. In some embodiments, subjects with PV with high HCT levels are at higher risk of developing thrombotic events (TE). In some embodiments, increased TE is associated with increased complications, such as cardiovascular complications, and increased morbidity and mortality in PV. In some embodiments, cytoreductive therapy, phlebotomy, and / or apheresis may be used to reduce excess red blood cells and lower HCT. HCT can be measured by any suitable known method in the art.Cytoreductive therapy refers to a certain medicine that is used to treat the reduction of blood cell level.Cytoreductive therapy includes interferon therapy, hydroxyurea, hydroxycarbamide, ruxolitinib, and / or anagrelide.

[0092] In some embodiments, subjects with PV-associated erythrocytosis have elevated hemoglobin (Hb) levels compared to subjects without PV. The normal range for Hb is about 13 g / dL to 17 g / dL in men and about 11 g / dL to 15 g / dL in women. In some embodiments, subjects with PV exhibit elevated Hb levels greater than 16.5 g / dL in men and greater than 16 g / dL in women. In some embodiments, the subject with PV has Hb level that is higher than 16g / dL, higher than 16.2g / dL, higher than 16.5g / dL, higher than 16.8g / dL, higher than 17g / dL, higher than 17.2g / dL, higher than 17.5g / dL, higher than 17.8g / dL, higher than 18g / dL, higher than 18.2g / dL, higher than 18.5g / dL, higher than 18.8g / dL, higher than 19g / dL, higher than 19.2g / dL, higher than 19.5g / dL, higher than 19.8g / dL, higher than 20g / dL or more.Hemoglobin level can be measured by any suitable known method in the art.

[0093] In some embodiments, a subject with PV-associated erythrocytosis exhibits trilineage cytosis in a bone marrow biopsy. In some embodiments, trilineage cytosis in the bone marrow manifests as a prominent erythroid, granulocytic, and megakaryocytic proliferation with pleomorphic, mature megakaryocytes (see, e.g., Mithoowani et al., Investigation and management of erythrocytosis, CMAJ. 2020 Aug 10; 192(32): E913-E918). Bone marrow cell morphology can be examined by any suitable method known in the art, e.g., bone marrow biopsy.

[0094] In some embodiments, a subject with PV-associated erythrocytosis may have a low serum erythropoietin (EPO) level (e.g., less than 3 mU / mL, less than 3 mU / mL, less than 2.9 mU / mL, less than 2.8 mU / mL, less than 2.7 mU / mL, less than 2.6 mU / mL, less than 2.5 mU / mL, less than 2.4 mU / mL, less than 2.3 mU / mL, less than 2.2 mU / mL, less than 2.1 mU / mL, or lower). Serum EPO can be measured by any suitable method known in the art.

[0095] In some embodiments, the subject has an increased red blood cell distribution width (RDW) compared to subjects without PV. The red blood cell distribution width (RDW) test measures the difference in volume and size of red blood cells (polycythemia). In some embodiments, a high RDW is associated with a higher risk of thrombosis in subjects with PV (see, e.g., Liu et al., RBC distribution width predicts thrombosis risk in polycythemia vera, Leukemia volume 36, pages 566-568 (2022)).

[0096] In some embodiments, the subject has leukocytosis compared to a subject without PV. Leukocytosis refers to a higher than normal white blood cell count. In some embodiments, a subject with PV exhibits leukocytosis in the absence of other factors that may cause an increase in white blood cell count (e.g., infection, acute inflammation).

[0097] In some embodiments, a subject with PV has splenomegaly. In PV, bone marrow produces excess red blood cells, white blood cells, and platelets, which can cause the spleen to become larger. In some embodiments, splenomegaly indicates disease progression in PV (Lee, et al., Volumetric Splenomegaly in Patients With Polycythemia Vera, J Korean Med Sci. 2022 Mar 21; 37(11): e87).

[0098] In some embodiments, patients with PV become iron deficient at the onset and / or during the course of the disease. However, the coexistence of iron deficiency and polycythemia results in a physiological discontinuity. In some embodiments, subjects with PV exhibit hepcidin suppression (i.e., low hepcidin levels) compared to subjects without PV. In some embodiments, hepcidin suppression in subjects with PV is due to increased erythropoietic activity. In some embodiments, increased erythropoiesis suppresses hepcidin through the erythropoietic hormone erythroferrone (ERFE) (Kautz et al., Identification of erythroferrone as an erythroid regulator of iron metabolism. Nat Genet. 2014;46(7):678-684). In some embodiments, hepcidin suppression in subjects with PV is due to iron deficiency. Under normal conditions, hepcidin inhibition mobilizes iron from hepatocyte stores, recycles it to splenic macrophages, and then exports it, increasing iron absorption by duodenal enterocytes and reversing iron deficiency. However, in some embodiments, hepcidin inhibition resulted in greater iron deficiency in PV subjects (e.g., PV subjects with one or more JAK2 mutations described herein) (see, e.g., Ginzburg et al., Dysregulated iron metabolism in polycythemia vera: etiology and consequences, Leukemia volume 32, pages 2105-2116 (2018)). In some embodiments, other conditions in PV, such as inflammation, counteract hepcidin inhibition. Abnormal hepcidin expression coupled with iron deficiency suggests that impaired iron metabolism is an important component of the pathology of PV. In some embodiments, subjects with PV have reduced serum hepcidin compared to subjects without PV.In some embodiments, subjects with PV have decreased serum prohepcidin compared to subjects without PV (Kwapisz et al., Decreased serum prohepcidin concentration in patients with polycythemia vera, J Zhejiang Univ Sci B. 2009 Nov; 10(11): 791-795). Hepcidin or prohepcidin can be measured by any suitable method known in the art, such as hepcidin prohormone ELISA, hepcidin ELISA, RT-PCR, etc. Although the exact mechanisms of hepcidin suppression and iron deficiency in PV patients remain unclear, in some embodiments, previous studies have shown that therapeutic agents that increase hepcidin levels in subjects with PV (e.g., ruxolitinib, a hepcidin mimetic) improve PV-related symptoms (e.g., lowering hematocrit (HCT), reducing splenomegaly) (see, e.g., Verstovsek et al., Markers of iron deficiency in patients with polycythemia vera receiving ruxolitinib or best available therapy. Leuk Res. 2017;56:52-59. May; Casu et al., Minihepcidin peptides as disease modifiers in mice affected by β-thalassemia and polycythemia vera. Blood. 2016;128:265-76).

[0099] In some embodiments, the occurrence of PV in a subject is associated with a mutation in a cell (e.g., a cell in the bone marrow, such as a hematopoietic progenitor cell). In some embodiments, the subject with PV has a mutation in the JAK2 gene (e.g., a JAK2 mutation that results in hyperactivation of JAK2 / STAT5) (see, e.g., Tefferi et al., Targeted Deep Sequencing in Polycythemia Vera and Essential Thrombocythemia. Blood Adv. 2016;1:21-30). In some embodiments, the subject with PV has one or more JAK2 mutations in the pseudokinase domain of the JAK2 gene (e.g., exons 12 to 15 of the JAK2 gene) (see, e.g., Lee et al., Structural Effects of Clinically Observed Mutations in JAK2 Exons 13-15: Comparison with V617F and Exon 12 Mutations. BMC Struct. Biol. 2009;9:58). In some embodiments, JAK2 mutations result in JAK2 hyperactivation, enhancing signaling through STAT5 (Kleppee et al., JAK-STAT Pathway Activation in Malignant and Nonmalignant Cells Contributes to MPN Pathogenesis and Therapeutic Response. Cancer Discov. 2015;5:316-331). Under normal conditions, JAK2 mediates signaling through the erythropoietin receptor (EpoR) upon binding of EPO to EpoR. EpoR / JAK2 activation triggers multiple signaling pathways that regulate the survival, proliferation, and differentiation of erythroid progenitors.In some embodiments, the JAK2 mutation results in EPO-independent JAK2 / STAT5 activation (James et al., A unique clonal JAK2 mutation leading to constitutive signaling causes polycythemia vera. Nature. 2005;434:1144-8; Baxter et al., Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet. 2005;365:1054-61; Akada et al., Conditional expression of heterozygous or homozygous Jak2V617F from its endogenous promoter induces a polycythemia vera-like disease. Blood. 2010;115:3589-97). In some embodiments, the EPO-independent JAK2 / STAT5 activation results in the continued proliferation and renewal of cells (e.g., hematopoietic progenitor cells) that have the mutation. In some embodiments, EPO-independent JAK2 / STAT5 activation results in erythrocytosis. In some embodiments, the subject with PV has a JAK2 exon 14 mutation. In some embodiments, the subject with PV has a JAK2 exon 14 mutation that causes JAK2 hyperactivation. In some embodiments, the subject with PV has one or more exon 14 mutations, including but not limited to, V617F, H606Q, H608Y, L611V, L611S, V617I, C618F, C618R, or absent exon 14 (see, e.g., Regimbeau et al., Genetic Background of Polycythemia Vera, Genes (Basel). 2022 Apr; 13(4): 637). In some embodiments, the subject has the JAK2 V617F mutation, which is the most common JAK2 mutation in PV patients. The V617F mutation is caused by a G>T point mutation in exon 14 of the JAK2 gene.The JAK2 V617F mutation contributes to destabilization of the inhibitory JH2-JH1 interface and / or activation of JH1, resulting in hyperactivation of JAK2 / STAT5 signaling (Constantinescu et al., Functional Consequences of Mutations in Myeloproliferative Neoplasms, Hemasphere. 2021 Jun 1;5(6):e578). In some embodiments, the allelic burden of the JAK2 V617F mutation influences the severity of PV. In some embodiments, a subject with PV is heterozygous for the JAK2 V617F mutation. In some embodiments, a subject with PV is homozygous for the JAK2 V617F mutation. In some embodiments, a JAK2 exon 14 mutation (e.g., a JAK2 V617F mutation) results in EPO-independent JAK2 / STAT5 hyperactivation. In some embodiments, subjects with PV who have a JAK2 exon 14 mutation (e.g., a JAK2 V617F mutation) have lower hepcidin levels compared to subjects without PV. In some embodiments, subjects with a JAK2 exon 14 mutation may have one or more other mutations described herein.

[0100] In some embodiments, subjects with PV have one or more JAK2 exon 12 mutations. In some embodiments, subjects with PV do not have a JAK2 V617F mutation. In some embodiments, JAK2 V617F mutation-negative PV subjects have one or more mutations in JAK2 exon 12. In some embodiments, JAK2 exon 12 mutations result in overactivation of the JAK2 / STAT5 pathway. In some embodiments, JAK2 exon 12 mutations result in erythrocytosis. In some embodiments, subjects with PV with JAK2 exon 12 mutations have similar serum and liver iron levels compared to subjects with PV with a JAK2 V617F mutation. In some embodiments, subjects with PV harboring a JAK2 exon 12 mutation have higher hemoglobin levels, higher ERFE levels, and lower hepcidin levels compared to subjects with PV harboring a JAK2 V617F mutation (see, e.g., Grisouard et al., JAK2 exon 12 mutant mice display isolated erythrocytosis and changes in iron metabolism favoring increased erythropoiesis. Blood. 2016;128:839-51). In some embodiments, subjects with PV harboring a JAK2 exon 12 mutation have a similar prognosis to subjects with PV harboring a JAK2 V617F mutation (Passamonti et al., Molecular and Clinical Features of the Myeloproliferative Neoplasm Associated with JAK2 Exon 12 Mutations. Blood. 2011;117:2813-2816).In some embodiments, the subject with PV has one of the following mutations: F537-K539delinsL, N542-E543del mutation, H538QK539L, V536-I546 dup11, V536-F547 and one or more exon 12 mutations, including, but not limited to, dup, F537-I546dup10F547L, F537IK539I, H538-K539delinsL, H538-K539del, H538DK539LI540S, H538G, K539L, K539E, I540-E543delinsMK, I540-E542delinsS, R541-E543delinsK, N542-E543del, D544-L545del, or 547insLI540-F547dup8 (see, e.g., Regimbeau et al., Genetic Background of Polycythemia Vera, Genes (Basel). 2022 Apr; 13(4): 637).

[0101] In some embodiments, subjects with PV have one or more JAK2 exon 13 mutations. In some embodiments, JAK2 exon 13 mutations result in hyperactivation of the JAK2 / STAT5 pathway. In some embodiments, JAK2 exon 13 mutations result in erythrocytosis. In some embodiments, subjects with PV have JAK2 exon 13 mutations and have lower hepcidin levels compared to subjects without PV. In some embodiments, subjects with PV have one or more exon 12 mutations, including but not limited to R564L, R564Q, V567A, G571S, G571R, L579F, H587N, S591L, or F557L (see, e.g., Regimbeau et al., Genetic Background of Polycythemia Vera, Genes (Basel). 2022 Apr; 13(4): 637).

[0102] In some embodiments, the subject with PV has a JAK2 exon 15 mutation. In some embodiments, the JAK2 exon 15 mutation results in overactivation of the JAK2 / STAT5 pathway. In some embodiments, the JAK2 exon 15 mutation results in erythrocytosis. In some embodiments, the subject with PV has a JAK2 exon 15 mutation that has a lower hepcidin level compared to a subject without PV. In some embodiments, the subject with PV has a I645V or L642P mutation in JAK2 exon 15 (see, e.g., Regimbeau et al., Genetic Background of Polycythemia Vera, Genes (Basel). 2022 Apr; 13(4): 637; Ma et al., Mutation Profile of JAK2 Transcripts in Patients with Chronic Myeloproliferative Neoplasias. J. Mol. Diagn. 2009; 11:49-53).

[0103] In some embodiments, a subject with PV has a non-JAK2 mutation that leads to overactivation of JAK2 / STAT5. For example, a subject with PV may have a mutation in the lymphocyte adaptor protein (LNK) gene (also known as SH2B adaptor protein 3 (SH2B3) gene). LNK is a negative regulator of JAK2 in cells (e.g., hematopoietic progenitor cells). In some embodiments, LNK can also modulate signaling mediated by lineage-specific cytokines (e.g., TPO and EPO), thereby controlling megakaryocyte and erythrocyte development, respectively. In some embodiments, LNK negatively modulates EPO receptor (EpoR) signaling by inhibiting a pathway (e.g., the JAK2 / STAT5 pathway) in primary erythroblasts. In some embodiments, the PV subject has a mutation in the LNK gene that results in loss of function of LNK, which leads to overactivation of the JAK2 / STAT5 pathway (see, e.g., Tefferi et al., Targeted Deep Sequencing in Polycythemia Vera and Essential Thrombocythemia. Blood Adv. 2016;1:21-30; McMullin et al., LNK Mutations and Myeloproliferative Disorders. Am. J. Hematol. 2016;91:248-251). In some embodiments, the PV subject with an LNK mutation does not have a JAK2 mutation. In some embodiments, the PV subject with an LNK mutation has a concurrent JAK2 mutation (e.g., a JAK2 V617F mutation).

[0104] In some embodiments, the subject with PV has a gene encoding ... The subject has one or more non-JAK2 mutations, including, but not limited to, the JAK2 gene, MPL gene, let-7a gene, miR-26b gene, miR-27b gene, miR-28 gene, miR-30b gene, miR-30c gene, miR-125-5p gene, miR-125b-5p gene, miR-143 gene, miR-145 gene, miR-150 gene, miR-182 gene, miR-223 gene, miR-342 gene, or miR-451 gene. In some embodiments, the non-JAK2 mutation may be present in a subject with a JAK2 mutation (e.g., JAK2 V617F or exon 12 mutation) (see, e.g., Regimbeau et al., Genetic Background of Polycythemia Vera, Genes (Basel). 2022 Apr; 13(4): 637).

[0105] In some embodiments, the mutation that results in JAK2 / STAT5 hyperactivation (e.g., any one of the mutations described herein) is an acquired mutation. In some embodiments, the mutation that results in JAK2 / STAT5 hyperactivation (e.g., any one of the mutations described herein) is a somatic mutation. In some embodiments, the subject with PV has acquired a mutation that causes JAK2 / STAT5 hyperactivation in cells within the bone marrow (e.g., any one of the mutations described herein). In some embodiments, the subject with PV has acquired a mutation that causes JAK2 / STAT5 hyperactivation in hematopoietic progenitor cells (e.g., any one of the mutations described herein). In some embodiments, the subject with PV has acquired CD34 + CD38 -The subject has acquired a mutation (e.g., any one of the mutations described herein) that causes hyperactivation of JAK2 / STAT5 in hematopoietic progenitor cells. In some embodiments, the subject with PV has acquired a mutation (e.g., any one of the mutations described herein) that causes hyperactivation of JAK2 / STAT5 in bone marrow progenitor cells. In some embodiments, the subject with PV has acquired a mutation (e.g., any one of the mutations described herein) that causes hyperactivation of JAK2 / STAT5 in megakaryocyte-erythroid progenitor cells (see, e.g., Jamieson et al., The JAK2 V617F mutation occurs in hematopoietic stem cells in polycythemia vera and predisposes toward erythroid differentiation, proc Natl Acad Sci U S A. 2006 Apr 18; 103(16): 6224-6229; Spivak, The polycythemia vera stem cell, Leukemia Suppl. 2014 Dec; 3(Suppl 1): S23-S24).

[0106] In some aspects, the present disclosure provides a method for treating polycythemia vera (PV) in a subject, wherein PV is associated with overactivation of the JAK2 / STAT5 pathway, and the method comprises administering to the subject an effective amount of an anti-TMPRSS6 antibody described herein. In some aspects, the present disclosure provides a method for treating polycythemia vera (PV) in a subject having bone marrow containing cells with JAK2 / STAT5 overactivation, the method comprises administering to the subject an effective amount of an anti-TMPRSS6 antibody described herein.

[0107] In some embodiments, administration of an anti-TMPRSS6 antibody described herein results in an increase in circulating hepcidin or prohepcidin levels (e.g., an increase in circulating hepcidin or prohepcidin levels by at least 5%, at least 10%, at least 20%, or at least 30%) in a subject with PV (e.g., a subject with PV associated with JAK2 / STAT5 hyperactivation) compared to the subject prior to receiving the anti-TMPRSS6 antibody or a subject with PV (e.g., a subject with PV associated with JAK2 / STAT5 hyperactivation) who has not received the anti-TMPRSS6 antibody. In some embodiments, administration of an anti-TMPRSS6 antibody described herein results in an increase in circulating hepcidin or prohepcidin levels (e.g., 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold). ...25%, at least 30%, at least 3 (Increase circulating hepcidin or prohepcidin levels by up to 5%, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95%, up to 100%, up to 1.5-fold, up to 2-fold, up to 3-fold, up to 4-fold, up to 5-fold, up to 10-fold, up to 20-fold, up to 50-fold, or up to 100-fold.) In some embodiments, increasing hepcidin in a subject with PV (e.g., a subject with PV associated with JAK2 / STAT5 hyperactivation) enhances iron-restricted erythropoiesis.

[0108] In some embodiments, administration of an anti-TMPRSS6 antibody results in a reduction in liver iron (e.g., a reduction in liver iron by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%) in a subject with PV (e.g., a subject with PV associated with JAK2 / STAT5 hyperactivation) compared to the subject before receiving the anti-TMPRSS6 antibody or a subject with PV but not receiving the anti-TMPRSS6 antibody (e.g., a subject with PV associated with JAK2 / STAT5 hyperactivation). Hepatic iron levels can be assessed by appropriate clinical tests, such as magnetic resonance imaging (MRI) (e.g., Henninger et al., Practical guide to quantification of hepatic iron with MRI, Eur Radiol. 2020; 30(1): 383-393), liver biopsy, etc.

[0109] In some embodiments, administration of an anti-TMPRSS6 antibody results in a reduction in circulating iron, such as serum iron, in a subject with PV (e.g., a subject with PV associated with JAK2 / STAT5 hyperactivation) compared to the subject before receiving the anti-TMPRSS6 antibody or a subject with PV (e.g., a subject with PV associated with JAK2 / STAT5 hyperactivation) who has not received the anti-TMPRSS6 antibody (e.g., a reduction in circulating iron by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%). Circulating iron levels can be assessed by appropriate clinical tests, such as measuring serum iron, transferrin saturation (TSAT), or total iron-binding capacity (TIBC).

[0110] In some embodiments, administration of an anti-TMPRSS6 antibody results in a reduction in hematocrit (HCT) (e.g., a reduction in HCT by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%) in a subject with PV (e.g., a subject with PV associated with JAK2 / STAT5 hyperactivation) compared to the subject before receiving the anti-TMPRSS6 antibody or a subject with PV (e.g., a subject with PV associated with JAK2 / STAT5 hyperactivation) who has not received the anti-TMPRSS6 antibody. Circulating iron levels can be assessed by appropriate clinical tests, such as magnetic resonance imaging (MRI) (e.g., Henninger et al., Practical guide to quantification of hepatic iron with MRI, Eur Radiol. 2020; 30(1): 383-393), liver biopsy, etc.

[0111] In some embodiments, administration of an anti-TMPRSS6 antibody results in a reduction in red blood cell count (e.g., a reduction in red blood cell count by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%) in a subject with PV (e.g., a subject with PV associated with JAK2 / STAT5 hyperactivation) compared to the subject before receiving the anti-TMPRSS6 antibody or a subject with PV but not receiving the anti-TMPRSS6 antibody (e.g., a subject with PV associated with JAK2 / STAT5 hyperactivation). Red blood cell count can be assessed by appropriate clinical tests, such as a blood smear, cell counter, or flow cytometry.

[0112] In some embodiments, administration of an anti-TMPRSS6 antibody results in a decrease in red blood cell distribution width (RDW) in a subject with PV (e.g., a subject with PV associated with JAK2 / STAT5 hyperactivation) compared to the subject before receiving the anti-TMPRSS6 antibody or a subject with PV (e.g., a subject with PV associated with JAK2 / STAT5 hyperactivation) who has not received the anti-TMPRSS6 antibody (e.g., a decrease in RDW by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%). RDW can be assessed by appropriate clinical tests, e.g., blood tests.

[0113] In some embodiments, administration of an anti-TMPRSS6 antibody results in a reduction in erythroid progenitor cells (e.g., a reduction in erythroid progenitor cells (e.g., in bone marrow) by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%) in a subject with PV (e.g., a subject with PV associated with JAK2 / STAT5 hyperactivation) compared to the subject before receiving the anti-TMPRSS6 antibody or a subject with PV but not receiving the anti-TMPRSS6 antibody (e.g., a subject with PV associated with JAK2 / STAT5 hyperactivation). Erythroid progenitor cells can be assessed by appropriate clinical tests, such as bone marrow biopsy, flow cytometry, etc.

[0114] In some embodiments, administration of an anti-TMPRSS6 antibody results in a reduction in plasma hemoglobin levels in a subject with PV (e.g., a subject with a PV associated with JAK2 / STAT5 hyperactivation) compared to the subject before receiving the anti-TMPRSS6 antibody or a subject with PV (e.g., a subject with a PV associated with JAK2 / STAT5 hyperactivation) who has not received the anti-TMPRSS6 antibody (e.g., a reduction in plasma hemoglobin levels by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%). Plasma hemoglobin levels can be assessed by appropriate clinical tests, such as blood tests.

[0115] In some embodiments, administration of an anti-TMPRSS6 antibody results in a decrease in mean corpuscular volume (MCV) in a subject with PV (e.g., a subject with PV associated with JAK2 / STAT5 hyperactivation) compared to the subject before receiving the anti-TMPRSS6 antibody or a subject with PV (e.g., a subject with PV associated with JAK2 / STAT5 hyperactivation) who has not received the anti-TMPRSS6 antibody (e.g., a decrease in MCV level by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%). MCV can be assessed by appropriate clinical tests, such as blood tests.

[0116] In some embodiments, administration of an anti-TMPRSS6 antibody results in a decrease in leukocytosis (i.e., white blood cell count) in a subject with a PV (e.g., a subject with a PV associated with JAK2 / STAT5 hyperactivation) compared to the subject before receiving the anti-TMPRSS6 antibody or a subject with a PV (e.g., a subject with a PV associated with JAK2 / STAT5 hyperactivation) who has not received the anti-TMPRSS6 antibody (e.g., a decrease in white blood cell count by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%). White blood cell count can be assessed by appropriate clinical tests, such as blood smears, cell counters, flow cytometry, etc.

[0117] In some embodiments, administration of an anti-TMPRSS6 antibody results in a reduction in thrombotic events (TE) (e.g., a reduction in TE by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%) in subjects with PV (e.g., subjects with PV associated with JAK2 / STAT5 hyperactivation) compared to subjects prior to receiving the anti-TMPRSS6 antibody or subjects with PV (e.g., subjects with PV associated with JAK2 / STAT5 hyperactivation) who have not received the anti-TMPRSS6 antibody. TE can be determined by appropriate clinical testing and clinic visits.

[0118] In some embodiments, administration of an anti-TMPRSS6 antibody results in a reduction in the frequency of phlebotomy in a subject with PV (e.g., a subject with a PV associated with JAK2 / STAT5 hyperactivation) compared to the subject before receiving the anti-TMPRSS6 antibody or a subject with PV (e.g., a subject with a PV associated with JAK2 / STAT5 hyperactivation) who has not received the anti-TMPRSS6 antibody (e.g., a reduction in the frequency of phlebotomy by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%). The need for phlebotomy can be determined by hematological evaluation of the subject, such as HCT, hemoglobin, red blood cell count, bone marrow biopsy, etc. In some embodiments, administration of an anti-TMPRSS6 antibody described herein results in 70% to 95% independence from phlebotomy (e.g., 70% to 95%, 75% to 90%, 80% to 85%, 85% to 90%) in treated subjects.

[0119] In some embodiments, administration of an anti-TMPRSS6 antibody results in a reduction in the frequency of cytoreductive therapy in a subject with PV (e.g., a subject with a PV associated with JAK2 / STAT5 hyperactivation) compared to the subject before receiving the anti-TMPRSS6 antibody or a subject with PV (e.g., a subject with a PV associated with JAK2 / STAT5 hyperactivation) who has not received the anti-TMPRSS6 antibody (e.g., a reduction in the frequency of cytoreductive therapy by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%). The need for cytoreductive therapy can be determined by hematological evaluation of the subject, such as HCT, hemoglobin, red blood cell count, bone marrow biopsy, etc.

[0120] In some embodiments, administration of an anti-TMPRSS6 antibody results in improved therapeutic benefit (e.g., reduction in HCT, hemoglobin, red blood cell count, improvement in iron deficiency, etc.) in subjects who have and are in need of PV but are refractory or intolerant to phlebotomy and / or cytoreductive therapy.

[0121] In some embodiments, the anti-TMPRSS6 antibody described herein can be administered to a subject in need thereof every week, every two weeks, every three weeks, every month, every two months, or every three months.In some embodiments, the anti-TMPRSS6 antibody is administered by intravenous injection, subcutaneous injection, or intraperitoneal injection.In some embodiments, the anti-TMPRSS6 antibody described herein can be administered in a clinic or can be self-administered by the subject (for example, by subcutaneous injection).

[0122] In some embodiments, administration of an anti-TMPRSS6 antibody results in a reduction from baseline in the Myeloproliferative Neoplasm Symptom Assessment Form (MPN-SAF). The MPN-SAF is a tool that allows MPN subjects (e.g., PV patients) to track their symptoms and monitor how they feel over time. Symptoms that may be monitored include, but are not limited to, fatigue, feeling full quickly when eating (early satiety), abdominal discomfort, lethargy, concentration problems, night sweats, itching (pruritus), bone pain, fever (over 100°F), and unintentional weight loss in the past six months. In some embodiments, more than 45% (e.g., more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, or 100%) of the subjects receiving anti-TMPRSS6 antibody treatment had a decrease of more than 50% (e.g., more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, or 100%) from each subject's baseline according to the MPN-SAF.

[0123] In some embodiments, administration of the anti-TMPRSS6 antibody has minor adverse effects on the treated subject, e.g., no hematological adverse events such as thrombocytopenia or neutropenia, no anti-drug antibodies.

[0124] In some embodiments, the present disclosure provides methods for treating a subject with PV by administering to the subject an effective amount of an anti-TMPRSS6 antibody in combination with any known therapeutic agent for treating PV, such as, for example, interferons (e.g., lopeginterferon a-2b-njft (Besremi), pegylated interferons), JAK2 inhibitors (e.g., ruxolitinib, XL019, fedratinib (SAR302503), momelotinib), JAK1 inhibitors (e.g., itacitinib), hepcidin mimetics (hepcidin memetic [e.g., rusfertide (PTG-300)], lysine-specific demethylase inhibitors [e.g., bomedemstat (IMG-7298)], TMPRSS6 antagonists [e.g., sapablursen (ISIS702843), SLN124], anti-TfR1 antibodies (e.g., PPMX-T003), MDM2 inhibitors [e.g., idasanutlin (RG7388), KRT-232], tyrosine kinase inhibitors [e.g., dasatinib, erlotinib, gleevec, lestaurtinib (CEP-701)], HDAC inhibitors [e.g., givinostat (ITF2357), MK-0683], PI3K inhibitors [e.g., umbralisib (TGR-1202)], telomerase inhibitors (e.g., imetelstat), phlebotomy, low-dose aspirin, or hydroxyurea.

[0125] Antibodies and antibody fragments that can alleviate one or more symptoms of a myeloproliferative disorder in vivo when administered to subjects representing animal models of the disease, i.e., subjects known to or suspected of having such a disorder, are expected to have therapeutic efficacy in clinical use. As demonstrated by the exemplary embodiment shown in Example 9 using the Jak2V617 / + Vav-iCre mouse model of PV, administration of the anti-TMPRSS6 recombinant monoclonal antibody MWTx-003 resulted in multiple in vivo effects, including, but not limited to, a dose-dependent decrease in hematocrit (HCT) levels, a reduction in circulating red blood cell (RBC) count, and hemoglobin (HGB) concentrations indicative of reduced erythrocytosis, as well as an increase in hepcidin levels, a decrease in serum iron concentration, and differential effects on the spleen and liver. Administration of the anti-TMPRSS6 recombinant monoclonal antibody MWTx-003 did not significantly change liver iron content, but caused a significant increase in iron deposition in splenic macrophages compared to isotype controls. Certain effects can be understood as an improvement in the symptoms of the disorder. The symptoms of the disorder are manifested in multiple biological systems, including, but not limited to, effects on the liver, spleen, blood (particularly serum hepcidin levels, RBC, HCT, erythrocytosis), and bone marrow. Administration of anti-TMPRSS6 antibodies improved multiple symptoms across disease model subjects, and measured symptom levels shifted from those seen in isotype controls of the disease model (untreated disease) to those seen in wild-type littermates, representing normal levels in genetically similar subjects not known or suspected to have the disease. Non-limiting exemplary embodiments of the present invention disclose anti-TMPRSS6 antibody therapy that increases hepcidin levels and reduces erythrocytosis in subjects with PV.

[0126] III. Composition Compositions are provided that comprise a safe and effective amount of the anti-TMPRSS6 antibody of the present invention and a pharmaceutically acceptable carrier or excipient suitable for the intended use of each composition.Such carriers include but are not limited to saline, buffer solution, glucose, water, glycerol, ethanol, excipients, stabilizers, preservatives or their combinations.It is understood that pharmaceutical preparations should be compatible with the mode of administration.

[0127] The anti-TMPRSS6 antibodies disclosed herein can be administered by any suitable means, including, but not limited to, injection or parenteral infusion. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, subcutaneous, or hepatic parenteral administration. The anti-TMPRSS6 antibodies disclosed herein can be formulated to be introduced into the liver tissue or vasculature for localized delivery to the target tissue. The anti-TMPRSS6 antibodies disclosed herein can be administered using a device, as a depot, or in a sustained-release preparation (e.g., a semipermeable matrix of a solid hydrophobic polymer containing the antibody, or a microcapsule) to enable slow and / or measured and / or localized delivery. The anti-TMPRSS6 antibodies disclosed herein can be formulated and administered using colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or macroemulsions.

[0128] IV. Method Methods are provided for treating iron metabolism disorders using an effective amount of the anti-TMPRSS6 antibody disclosed herein. Without wishing to be bound by a particular mechanism of action, methods are provided for targeting TMPRSS6 using the anti-TMPRSS6 antibody disclosed herein, which result in multiple downstream effects, particularly effects on components (molecules, systems, processes) involved in iron metabolism and erythropoiesis. Without wishing to be bound by a particular mechanism of action, methods are provided for treating iron metabolism disorders using an effective amount of the anti-TMPRSS6 antibody disclosed herein to modulate the activity of components involved in iron metabolism. In particular, methods are provided for treating iron overload associated with excessive iron accumulation in tissues and organs, including disorders associated with or characterized by ineffective erythropoiesis, which may include, but are not limited to, β-thalassemia, particularly non-transfusion-dependent thalassemia, MDS (myelodysplastic syndrome), dyserythropoietic anemia, and sideroblastic anemia. Without being limited to a single mechanism of action, methods are provided for treating disorders associated with suppression of hepcidin expression, including iron overload associated with low hepcidin levels, particularly diseases or conditions involving abnormal suppression of hepcidin expression, by administering an anti-TMPRSS6 antibody that can increase hepcidin expression. Methods are provided for treating myeloproliferative disorders. Methods are provided for treating polycythemia vera (PV). Methods are provided for treating polycythemia vera (PV) associated with insufficient hepcidin suppression.

[0129] Provided herein is a method for treating a disorder of iron metabolism, comprising administering an effective amount of an anti-TMPRSS6 antibody disclosed herein to a subject in need thereof, wherein administering the effective amount of the anti-TMPRSS6 antibody ameliorates at least one biological effect (symptom) associated with the disorder. Provided is a method for treating a disorder of iron metabolism associated with depressed hepcidin levels, wherein administering an effective amount of an anti-TMPRSS6 antibody disclosed herein to a subject in need thereof results in at least one of increased hepcidin promoter activity, increased hepcidin transcription, increased hepcidin RNA levels, and increased hepcidin levels, particularly serum hepcidin levels. Methods are provided for treating a subject known to have or suspected of having iron overload, wherein administration of an effective amount of an anti-TMPRSS6 antibody results in one or more biological effects, including, but not limited to, reducing liver non-heme iron, increasing serum hepcidin, increasing liver hepcidin RNA, reducing splenomegaly, increasing red blood cell count (RBC), increasing hematocrit (HCT), reducing red blood cell distribution width (RDW), and increasing production of mature red blood cells (increased erythropoiesis). Methods are provided for treating a subject known to have or suspected of having iron overload characterized by ineffective erythropoiesis, wherein administration of an effective amount of an anti-TMPRSS6 antibody results in one or more biological effects, including, but not limited to, reducing hepatic non-heme iron, increasing serum hepcidin, increasing liver hepcidin RNA, reducing splenomegaly, increasing red blood cell count (RBC), increasing hematocrit (HCT), reducing red blood cell distribution width (RDW), and increasing production of mature red blood cells (increased erythropoiesis).

[0130] Methods and compositions are provided for treating disorders of iron metabolism, particularly iron overload, and more particularly iron overload characterized by ineffective erythropoiesis, in which administration of an effective amount of an anti-TMPRSS6 antibody results in treatment or amelioration of two or more biological effects or symptoms associated with the disorder. Without wishing to be bound by theory or mechanism of action, it is understood that ineffective erythropoiesis, characterized by low production of mature red blood cells in the bone marrow due to apoptosis of erythroid precursors, is the driving force behind abnormal hepcidin suppression, leading to increased iron absorption and iron overload. Based on this understanding, treatments that improve erythroblast differentiation and maturation into red blood cells should be therapeutically beneficial for treating iron overload. The effectiveness of anti-TMPRSS6 antibody therapy, such as increasing erythroblast differentiation and maturation into red blood cells, reducing iron load, and increasing hepcidin expression, maximizes the therapeutic benefits of methods and compositions using the anti-TMPRSS6 antibodies disclosed herein.

[0131] Methods and compositions are provided for treating myeloproliferative disorders, particularly myeloproliferative neoplasms such as chronic myeloproliferative neoplasms, more particularly myeloproliferative neoplasms characterized by erythroid hyperplasia, and even more particularly polycythemia vera (PV), in which administration of an effective amount of an anti-TMPRSS6 antibody treats or ameliorates two or more biological effects or symptoms associated with the disorder. While not wishing to be bound by theory or mechanism of action, given the degree of iron deficiency observed in PV patients, the observation of insufficient suppression of hepcidin in PV patients is understood to suggest that impaired or dysregulated iron metabolism is an important component of the pathology of PV, and in particular, insufficient suppression of hepcidin levels is a component of the pathology of PV. Based on this understanding, treatments that modulate hepcidin expression should be therapeutically beneficial for the treatment of myeloproliferative neoplasms characterized by erythroid hyperplasia, particularly polycythemia vera (PV). The efficacy of anti-TMPRSS6 antibody therapy in reducing erythrocytosis, normalizing hematocrit (HCT) levels, and increasing hepcidin expression, among other things, maximizes the therapeutic benefit of the methods and compositions using anti-TMPRSS6 antibodies disclosed herein.

[0132] The following examples are offered to illustrate, but not to limit, the claimed invention. [Example]

[0133] [Example 1] Antibody production and identification of antibodies that bind to TMPRSS6 The production of a novel monoclonal antibody against TMPRSS6 was performed under contract by the LakePharma Discovery Immunology group (LakePharma, Inc., San Carlos, CA) using in vivo rodent immunization and hybridoma technology. A mixture of pLEV113_huTMPRSS6 and pLEV113_moTMPRSS6-TCE plasmid DNA (cloned at LakePharma, Inc.) was used for DNA-based immunization in B6;SJL mice (The Jackson Laboratories) via hydrodynamic tail vein injection. Sufficient plasma titers, as determined by fluorescence-activated cell sorting (FACS), were obtained to facilitate downstream antibody recovery and screening activities. Electrofusion was performed using pooled spleen cells from two immunized mice and their myeloma fusion partners using a NEPA GENE ECFG21 Super Electro Cell Fusion Generator (Nepa Gene Co., Ltd., Ichikawa-City, Chiba, Japan). The fusion material was plated into ten 384-well plates in low-xanthine-aminopterin-thymidine medium, which specifically selects hybridomas over unfused myeloma partner cells. Hybridoma supernatants were initially screened for HuTMPRSS6 reactivity by FACS analysis to detect supernatants that showed positive staining signals on TMPRSS6-expressing HEK293T cells (HEK293T cells were transfected with a plasmid encoding huTMPRSS6-(His)6 (SEQ ID NO: N97) and negative staining on the parental cells (HEK293T) 10 days after fusion. Hybridoma supernatants that showed positive staining signals on TMPRSS6-expressing HEK293 cells and negative staining on the parental cells were designated "hits" for further screening. 192 hits were identified in the primary FACS screen, and 143 hits were confirmed in secondary and tertiary FACS screens.

[0134] Example 2: Functional screening of anti-TMPRSS6 antibodies; identification, generation, and sequencing of monoclonal anti-TMPRSS6 antibodies and humanized variants HAMP-luciferase reporter assay A hepcidin promoter-luciferase reporter assay was used to measure the response of the HAMP promoter to various anti-TMPRSS6 antibodies (Du, X. et al., 2008. Science 320: 1088-1092; modified to use the human HAMP promoter instead of the originally disclosed mouse HAMP promoter). For the HAMP-luciferase reporter assay, a 2.5 kb HAMP promoter fragment (reference genome GRCh38) was spliced ​​upstream of the sequence encoding firefly luciferase. A control construct encoding Renilla luciferase driven by a thymidine kinase promoter (Promega, E6931) was used as an internal control. These constructs were co-transfected with a construct encoding TMPRSS6 into HepG2 cells (ATCC, HB-8065). Transfected HepG2 cells expressing TMPRSS6 were pretreated for approximately 3 hours with various concentrations of purified mAb diluted in starvation medium containing Minimum Essential Medium (MEM, ATCC) + 1% heat-inactivated fetal bovine serum (FBS, Gibco) + 1 mM sodium pyruvate + non-essential amino acid solution (Gibco) + 10 mM HEPES (Gibco) + 1% Pen / Strep (Gibco). Then, they were treated with recombinant hBMP6 (R&D Systems) at a final concentration of 25–60 ng / ml to induce BMP-SMAD-mediated signaling. Purified mouse IgG (Sigma-Aldrich) or human IgG1 (BioXcell) was used as a control. After overnight hBMP6 treatment, cells were lysed and luciferase substrate was added. Luminescence readings for firefly luciferase and Renilla luciferase were recorded by measuring total light output, respectively. Activity was calculated as the ratio of firefly luciferase luminescence to Renilla luciferase luminescence (control). The results of these assays are shown in Figures 2A-2F.

[0135] In vitro functional screening To screen for functionally active hybridomas, all 143 HuTMPRSS6-binding hybridomas ("hits") were tested using the HAMP-luciferase reporter assay described above. Supernatants from 10 of the 143 HuTMPRSS6-binding hybridomas increased HAMP promoter activity (data not shown) and were identified as "active clones" for further testing. These 10 active clones were tested for cross-reactivity to the mouse target MoTMPRSS6, as described in Example 4 below, and three showed binding to both HuTMPRSS6 and MoTMPRSS6, as measured by FACS. These three cross-reactive clones were further plated at a density of 1 cell / well in 192 wells of a 384-well plate to generate monoclonal hybridoma clones. Among the resulting subclones, those that exhibited the desired functional activity and cross-reactivity to non-human targets, i.e., mouse TMPRSS6 (moTMPRSS6) and / or cynomolgus monkey TMPRSS6 (cynoTMPRSS6), were identified as MWTx-001, MWTx-002, and MWTx-003.

[0136] Sequences of anti-TMPRSS6 antibodies MWTx-001, MWTx-002, and MWTx-003 The sequences of MWTx-001, MWTx-002, and MWTx-003 were determined by isolating mRNA from each hybridoma sample and performing reverse transcription-polymerase chain reaction (RT-PCR) using a unique mouse IgG-specific primer set to amplify the target variable regions for sequencing. Unique heavy and light chains were identified for each anti-TMPRSS6 antibody. The nucleotide sequences of each heavy and light chain were determined. The amino acid sequences encoded by the nucleotide sequences were determined, and the CDR regions were identified using the Kabat numbering system. Table 1 shows the amino acid sequences of the heavy and light chain variable regions, as well as the amino acid sequences of the identified CDRs (based on Kabat numbering), and the nucleotide sequences of the heavy and light chain variable regions for MWTx-001, MWTx-002, and MWTx-003, respectively.

[0137] [Table 1-1] [Table 1-2] [Table 1-3]

[0138] Generation and screening of humanized anti-TMPRSS6 antibody variants The parent antibody was humanized using CDR grafting onto a human antibody framework. First, homology modeling of the parent antibody's 3D structure was performed to establish a structural model. The amino acid sequences of the variable fragment framework were identified based on overall sequence identity, matching positions at the VH-VL interface, canonical positions of similarly classified CDRs, and removal of potential N-glycosylation sites. Humanized antibodies were designed by creating multiple hybrid sequences in which selected portions of the parent antibody sequence were fused with human framework sequences. The isotypes selected for formulating the humanized antibodies were IgG1 for the heavy chain and IgG1 kappa for the light chain. Using the 3D model, these humanized sequences were methodologically analyzed by eye and computer modeling to isolate sequences most likely to retain antigen binding. The goal was to maximize the amount of human sequence in the final humanized antibody while maintaining the specificity of the original antibody. Humanized variants were expressed as humanized VH and VL pairs and purified for affinity analysis.

[0139] In one round of designing, generating, and testing variants as part of affinity analysis, four VH variants were generated with the VH-CDRs of the parent antibody MWTX-003 in corresponding positions on four different human IgG1-derived frameworks (SEQ ID NOS: 89-92), and four VL (VK) variants were generated with the VL-CDRs of the parent antibody MWTX-003 in corresponding positions on four different human IgG1 kappa-derived frameworks (SEQ ID NOS: 93-96). A total of 16 humanized variants representing every combination of VH and VL (VK) variants were prepared according to a 4VH x 4VK matrix, and the antigen-binding properties (k on , k off , KD) was evaluated and found to have a KD value in the nanomolar range of 4.16E-07 (~1.09E-08).

[0140] Mutants that exhibited desirable antigen-binding affinities were selected for further evaluation and development. In some cases, the parent CDR sequences were modified to avoid potentially undesirable events, such as aspartic acid isomerization.

[0141] To suppress antibody effector functions, particularly antibody-dependent cellular cytotoxicity (ADCC), key amino acid residues in the Fc region were identified and mutated (substituted) for all humanized antibody variants. Using the guidance available in the published literature regarding Fc mutations to achieve the goal of abrogating ADCC, the mutations of the present invention, for example, the native Fc in hIgG1, as described in (Tamm A, Schmidt RE. IgG binding sites on human Fc gamma receptors. Int Rev Immunol. 1997;16(1-2):57-85. Doi: 10.3109 / 08830189709045703; Jefferis R, Lund J. Interaction sites on human IgG-Fc for Fc gamma R: current models. Immunol Lett. 2002 Jun 3;82(1-2):57-65. Doi: 10.1016 / s0165-2478(02)00019-6). This information is provided for the removal of the N-linked glycosylation site (N297A mutation) or the substitution of leucines at positions 234 and 235 of the smaller hinge region in the Fc (LALA double mutation). In the present variants, the N297A mutation was introduced into the Fc of the hzMWTx-001Var and hzMWTx-002Var antibodies, and the LALA mutation was introduced into the Fc of the hzMWTx-003Var antibody to achieve the same goal of reducing or silencing ADCC (Table 3, SEQ ID NOs: 73, 77, 81).

[0142] As a result of the evaluation, the humanized anti-TMPRSS6 antibody variants hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var were selected for further testing. The sequences and characteristics of the humanized variants are shown in Tables 2 and 3 below.

[0143] Recombinant production of humanized anti-TMPRSS6 antibody variants Expression constructs for humanized anti-TMPRSS6 antibody variants were engineered to contain an internal ribosome entry site (IRES) between the LC- and HC-encoding DNA sequences, codon-optimized by Geneart DNA synthesis, and cloned into the pcDNA3.4 mammalian expression vector (ThermoFisher). The sequence of the DNA insert was confirmed by sequencing. For recombinant antibody production, the expression construct was used for transient transfection using the ExpiCHO expression system (ThermoFisher) according to the manufacturer's instructions. The expressed antibody was purified by Protein A affinity chromatography. The antibody yields from transient transfection ranged from 50 mg to 300 mg per liter, with purity exceeding 95% and endotoxin levels below 1 EU / ml.

[0144] Sequences of humanized anti-TMPRSS6 antibody variants hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var

[0145] Humanized anti-TMPRSS6 antibody variants hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var were selected for further testing. The variable region sequences for each are shown in Table 2 below, with identified CDRs underlined and changes made in the humanized variant CDR sequences relative to the parent antibody indicated and discussed.

[0146] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0147] Table 3 shows the complete heavy and light chain protein and nucleotide sequences of the anti-TMPRSS6 monoclonal antibodies MWTx-001, MWTx-002, and MWTx-003, and the humanized anti-TMPRSS6 antibody variants hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var. The heavy chain protein sequences of the humanized anti-TMPRSS6 antibody variants hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var indicate the positions of the mutations (changes) introduced to reduce ADCC as described above.

[0148] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9]

[0149] Dose-dependent effect of anti-TMPRSS6 antibody on HAMP promoter activity Figures 2A-2F show the results of using the above-described HAMP-luciferase reporter assay to test MWTx-001, MWTx-002, MWTx-003, and their humanized variants hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var at the indicated concentrations. MWTx-001 (Figure 2A), MWTx-002 (Figure 2B), MWTx-003 (Figure 2C), and their humanized variants hzMWTx-001Var (Figure 2D), hzMWTx-002Var (Figure 2E), and hzMWTx-003Var (Figure 2F) each increase HAMP promoter activity in a dose-dependent manner. The EC of MWTx-001 50 The EC value of MWTx-002 was calculated to be 3 μg / ml (Figure 2A). 50 The EC value of MWTx-003 was calculated to be 1 μg / ml (Figure 2B). 50 The EC value of hzMWTx-001Var was calculated to be 2 μg / ml (Figure 2C). 50 The EC value of hzMWTx-002Var was calculated to be 0.8 μg / ml (Figure 2D). 50 The EC value of hzMWTx-003Var was calculated to be 0.3 μg / ml (Figure 2E). 50 was calculated to be 0.3 μg / ml (Fig. 2F).

[0150] [Example 3] Binding affinity of anti-TMPRSS6 antibodies The binding affinity of various anti-TMPRSS6 antibodies to human TMPRSS6 expressed on HEK293T cells was measured using three different methods: cell surface ELISA (Figures 3A–3C), FACS (Figures 3D–3F), and biolayer interferometry (Figures 3G–3M).

[0151] Binding affinity measurement of anti-TMPRSS6 mAb using cell surface ELISA HEK293T cells stably expressing human TMPRSS6 (generated by LakePharma Inc. as described above; SEQ ID NO: 97) were fixed with 4% paraformaldehyde (PFA), washed with dPBS (Dulbecco's Phosphate-Buffered Saline, Corning Cellgro), and then incubated with various concentrations of anti-TMPRSS6 antibody diluted in BSA medium [DMEM + 1% Pen / Strep + 10 mM HEPES + 1 mg / ml BSA (Sigma-Aldrich)]. Purified mouse IgG was used as a control (Sigma-Aldrich). After incubation, cells were washed with BSA medium and incubated with HRP-conjugated goat anti-mouse IgG (Invitrogen) as the secondary antibody. Finally, cells were washed with dPBS to remove unbound antibody and developed with ELISA liquid substrate (Sigma-Aldrich). The reaction was stopped by adding an equal volume of 1 M H2SO4 ELISA liquid substrate. Bound antibody was detected at OD 450nm The results of these assays are shown in Figures 3A-3C.

[0152] Binding affinity measurement of anti-TMPRSS6 mAb using FACS HEK293T cells stably expressing human TMPRSS6 were harvested and blocked with dPBS + 3% BSA before incubation with various concentrations of anti-TMPRSS6 antibodies diluted in dPBS + 3% BSA. Purified mouse IgG was used as a control. After incubation, cells were washed with dPBS and incubated with APC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Inc.) as the secondary antibody. Finally, cells were washed with dPBS to remove unbound antibody, resuspended in dPBS + 1 mM EDTA, and then subjected to FACS analysis using a NOVOCYTE® flow cytometer (ACEA Biosciences, Inc., San Diego, CA). Bound antibody was determined by measuring the mean APC intensity after excitation at 640 nm and emission (fluorescence) at 675 nm. The results of these assays are shown in Figures 3D-3F.

[0153] Determining the affinity and binding kinetics of anti-TMPRSS6 antibodies using biolayer interference Biolayer interferometry was used to measure the affinity and determine the binding kinetics of anti-TMPRSS6 antibodies using the Octet RED96e system (Sartorius AG). Prehydrated anti-mouse IgG Fc capture (AMC) biosensors (MWTx-001, MWTx-002, and MWTx-003 anti-TMPRSS6 antibodies, Figures 3G–3I) or anti-anti-human IgG Fc capture (AHC) biosensors (hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var anti-TMPRSS6 antibodies, Figures 3J–3L) were first immersed in 1x KB (Kinetic Buffer, 1x PBS pH 7.0). After equilibration for 120 seconds with 7.4 + 0.02% Tween-20 + 0.1% BSA to establish a first baseline, 10 mg / ml of anti-TMPRSS6 antibody (MWTx-001, Figure 3G; MWTx-002, Figure 3H; MWTx-003, Figure 3I; hzMWTx-001Var, Figure 3J; hzMWTx-002Var, Figure 3K; hzMWTx-003Var, Figure 3L) was loaded onto the AMC or AHC biosensor for 240 seconds. Next, a second baseline signal was established for 120 seconds, followed by association with various concentrations of human ecto-TMPRSS6-FLAG (SEQ ID NO: 102) (generated in-house by fusing the extracellular domain of human TMPRSS6 with a C-terminal FLAG tag). Finally, the analyte was dissociated with 1x KB for 360 seconds. Data analysis was performed using Octet Data Analysis HT Software. on , k off and R 2 are summarized in Figure 3M.

[0154] [Example 4] Cross-reactivity: Binding of anti-TMPRSS6 antibodies to human TMPRSS6 and non-human TMPRSS6 Determination of cross-reactivity by FACS The selected anti-TMPRSS6 antibodies were tested to determine whether they could bind to TMPRSS6 derived from mouse and / or cynomolgus monkeys. HEK293T cells stably expressing human TMPRSS6 [HuTMPRSS6-(His)6] (generated by LakePharma Inc. as described above), HEK293T cells stably expressing mouse TMPRSS6 [MoTMPRSS6-(His)6] (SEQ ID NO: 98) (generated by LakePharma Inc. as described above), and HEK293T cells transiently expressing cynomolgus monkey TMPRSS6 [CynoTMPRSS6-(His)6] (SEQ ID NO: 99) were collected. HEK293T cells stably expressing human TMPRSS6 were used as a positive control, and HEK293T cells were used as a negative control (as described above). Cells were blocked with dPBS + 3% BSA before incubation with anti-TMPRSS6 antibodies diluted in dPBS + 3% BSA. After incubation, cells were washed with dPBS and further incubated with AlexaFluor-488-conjugated goat anti-mouse IgG (Invitrogen) as the secondary antibody. Finally, cells were washed with dPBS to remove unbound antibody, resuspended in dPBS + 1 mM EDTA, and then subjected to FACS analysis using a NOVOCYTE® flow cytometer (ACEA Biosciences, Inc., San Diego, CA). Bound antibody was determined by excitation at 488 nm and measuring emission (FITC-A) at 530 nm. The results of these assays are shown in histogram plots in Figures 4A–4I. Cross-reactivity with mouse TMPRSS6 was observed with MWTx-001 (Figure 4D) and MWTx-003 (Figure 4F), whereas MWTx-002 (Figure 4E) showed no detectable cross-reactivity with mouse TMPRSS6. Cross-reactivity with cynomolgus monkey TMPRSS6 was observed for MWTx-001 (4G), MWTx-002 (Fig. 4H), and MWTx-003 (Fig. 4I).

[0155] Determination of cross-reactivity by cell surface ELISA HEK293T cells stably expressing mouse TMPRSS6 (generated by LakePharma Inc. as described above; Figures 4J, 4L, 4N, 4P, 4R, 4T) or cynomolgus monkey (generated in-house as described above; Figures 4K, 4M, 4O, 4Q, 4S, 4U) were fixed with methanol (100%), washed with dPBS (Dulbecco's Phosphate-Buffered Saline, Corning Cellgro) and then incubated with various concentrations of anti-TMPRSS6 antibodies and their humanized variants diluted in BSA medium [DMEM + 1% Pen / Strep + 10 mM HEPES + 1 mg / ml BSA (Sigma-Aldrich)]. Purified mouse IgG (Figures 4J-4O) or human IgG1 (Figures 4P-4U) were used as controls. After incubation, the cells were washed with BSA medium and incubated with HRP-conjugated goat anti-mouse (Invitrogen, Figure 4J-4O) or anti-human (Millipore, Figure 4P-4U) IgG as the secondary antibody. Finally, the cells were washed with dPBS to remove unbound antibodies and developed with ELISA liquid substrate (Sigma-Aldrich). The reaction was then stopped by adding an equal volume of 1 M H2SO4 ELISA liquid substrate. Bound antibodies were detected at OD 450nm The results of these assays are shown in Figures 4J–4U. Cross-reactivity with mouse TMPRSS6 was observed with the MWTx-001 (Figure 4J) and MWTx-003 (Figure 4N) anti-TMPRSS6 antibodies and their humanized variants, hzMWTx-001Var (Figure 4P) and hzMWTx-003Var (Figure 4T), whereas the MWTx-002 (Figure 4L) anti-TMPRSS6 antibody or its humanized variant, hzMWTx-002Var (Figure 4R), showed no detectable cross-reactivity with mouse TMPRSS6. Cross-reactivity with cynomolgus monkey TMPRSS6 was observed for the MWTx-001 (Fig. 4K), MWTx-002 (Fig. 4M), and MWTx-003 (Fig. 4O) anti-TMPRSS6 antibodies and their humanized variants, hzMWTx-001Var (Fig. 4Q), hzMWTx-002Var (Fig. 4S), and hzMWTx-003Var (Fig. 4U) anti-TMPRSS6 antibodies.

[0156] [Example 5] Target specificity: Binding of anti-TMPRSS6 antibodies to homologous matriptase. To determine whether anti-TMPRSS6 antibodies bind to homologous matriptase, HEK293T cells overexpressing matriptase (ST14) (SEQ ID NO: 100) (Figures 5B, 5E, 5H, 5K, 5N, 5Q) and HEK293T cells overexpressing matriptase-3 (TMPRSS7) (SEQ ID NO: 101) (Figures 5C, 5F, 5I, 5L, 5O, 5R) were collected (generated in-house). HEK293T cells stably expressing human TMPRSS6 (matriptase-2) (SEQ ID NO: 97) (generated by LakePharma Inc., as described above, Figures 5A, 5D, 5G, 5J, 5M, 5P) were used as a positive control, and HEK293T cells (Figures 5A-5R) were used as a negative control (as described above). Cells were blocked and permeabilized with dPBS + 3% BSA + 0.1% Tween-20 and then incubated with various anti-TMPRSS6 antibodies diluted in dPBS + 3% BSA + 0.1% Tween-20. Cells were incubated with anti-TMPRSS6 antibodies and their humanized variants at approximately 1 μg / ml for 1 hour. After incubation, cells were washed with dPBS and incubated with AlexaFluor-488-conjugated goat anti-mouse IgG (Invitrogen, Figures 5A-5I) or Allophycocyanin (APC)-conjugated goat anti-human IgG (Jackson Immuno Research, Figures 5J-5R) as secondary antibodies. Finally, cells were washed with dPBS, resuspended in dPBS + 1 mM EDTA, and then subjected to FACS analysis using a NOVOCYTE® flow cytometer. Bound antibodies were determined by excitation at 488 nm and measuring emission at 530 nm (FITC-A) (Figures 5A-5I) or by excitation at 640 nm and measuring emission at 675 nm (APC-A) (Figures 5J-5R). The results of these assays are shown in histogram plots in Figures 5A-5R. All antibodies showed binding to human TMPRSS6 (matriptase-2) (Figures 5A, 5D, 5G, 5J, 5M, 5P), and none of the antibodies showed binding to the homologous matriptase ST14 (Figures 5B, 5E, 5H, 5K, 5N, 5Q) or TMPRSS7 (Figures 5C, 5F, 5I, 5L, 5O, 5R).The MWTx-001 anti-TMPRSS6 antibody and its humanized variant, hzMWTx-001Var anti-TMPRSS6 antibody, showed binding to human TMPRSS6 (Figures 5A and 5J), but not to matriptase (ST14) (Figures 5B and 5K) or matriptase-3 (TMPRSS7) (Figures 5C and 5L). The MWTx-002 anti-TMPRSS6 antibody and its humanized variant, hzMWTx-002Var anti-TMPRSS6 antibody, showed binding to human TMPRSS6 (matriptase-2) (Figures 5D and 5M), but not to matriptase (ST14) (Figures 5E and 5N) or matriptase-3 (TMPRSS7) (Figures 5F and 5O). The MWTx-003 anti-TMPRSS6 antibody and its humanized variant hzMWTx-003Var anti-TMPRSS6 antibody showed binding to human TMPRSS6 (matriptase-2) (Figures 5G, 5P), but not to matriptase (ST14) (Figures 5H, 5Q) or matriptase-3 (TMPRSS7) (Figures 5I, 5R).

[0157] Example 6: Treatment with anti-TMPRSS6 antibody in a mouse pharmacodynamic model To test the in vivo pharmacodynamic response of anti-TMPRSS6 antibodies, 2–10 mg / kg of MWTx-003 anti-TMPRSS6 antibody (Figures 6A–6B, 6D–6E, 6G–6H, 6J–6K) or its humanized variant hzMWTx-003Var anti-TMPRSS6 antibody (Figures 6C, 6F, 6I, 6L) was intraperitoneally injected into wild-type C57BL / 6J mice. Mouse IgG2b (BioXcell, Figures 6A–6B, 6D–6E, 6G–6H, 6J–6K) or human IgG1 (BioXcell, Figures 6C, 6F, 6I, 6L) was used as an isotype control. Twenty hours after injection, 50 µg of GFP-TMPRSS6 plasmid DNA (generated in-house by inserting human TMPRSS6 into a GFP vector) was delivered to each mouse via hydrodynamic tail vein injection. Mice were euthanized 44 hours after hydrodynamic injection, and liver tissue and blood were collected. Liver RNA was purified using EZgene Total RNA Purification Plus from Biomiga (San Diego, CA) according to the manufacturer's instructions. Mouse serum was obtained by centrifugation of whole blood at 1500 × g for 10 minutes.

[0158] Effect of treatment with anti-TMPRSS6 antibody on serum iron Serum iron was measured by a chromogenic assay developed in-house (Figures 6A-6C). Briefly, mouse serum or iron standards (31-500 μg / dL) were mixed with Mixed Acid Solution (0.6 M trichloroacetic acid, 0.4 M sodium thioglycolate, 1 M hydrochloric acid) by vortexing for 30 seconds. The mixture was incubated at 37°C for 10 minutes, then centrifuged at 10,000 × g for 10 minutes and developed with a color development solution (1.5 M sodium acetate, 0.5 mM bathophenanthroline disulfonate). The absorbance was then measured at OD . 535nm Serum iron concentrations were calculated from a linear iron standard curve. Treatment with 10 mg / kg of the MWTx-003 anti-TMPRSS6 antibody (Figures 6A-6B) and its humanized variant, hzMWTx-003Var anti-TMPRSS6 antibody (Figure 6C), significantly reduced serum iron.

[0159] Effect of treatment with anti-TMPRSS6 antibody on serum hepcidin Serum hepcidin was measured using a Hepcidin-Murine Compete ELISA kit purchased from Intrinsic Lifesciences (La Jolla, CA) according to the manufacturer's instructions (Figures 6D-6F). Briefly, diluted mouse serum or hepcidin standards were mixed with hepcidin-biotin conjugate and then added to plates coated with anti-mouse hepcidin antibody. Serum hepcidin or hepcidin standards compete with the hepcidin-biotin conjugate for binding to the coated anti-hepcidin antibody. Bound hepcidin-biotin conjugate was detected with streptavidin-conjugated horseradish peroxidase (HRP), developed with TMB, followed by stop solution. Absorbance was then measured at OD . 450nm Data were analyzed using a four-parameter logistic (4-PL) curve fit in Graphpad Prism 8, and serum hepcidin concentrations were interpolated. Hydrodynamic delivery of GFP-TMPRSS6 significantly reduced serum hepcidin levels (Figure 6D), whereas treatment with 10 mg / kg of the MWTx-003 anti-TMPRSS6 antibody (Figures 6D-6E) and its humanized variant, hzMWTx-003Var anti-TMPRSS6 antibody (Figure 6F), reversed the suppression of hepcidin and significantly increased serum hepcidin levels.

[0160] Effect of treatment with anti-TMPRSS6 antibody on hepatic hepcidin RNA Liver hepcidin RNA was quantified by real-time qPCR (Figures 6G–6I). Briefly, cDNA was first synthesized from liver RNA using iScript Reverse Transcription Supermix (Bio-Rad) according to the manufacturer's instructions. Hepcidin transcripts were amplified with the specific primers shown below and detected using SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad) on a Bio-Rad CFX96 qPCR instrument according to the manufacturer's instructions. Samples were analyzed in triplicate, and results were normalized to β-actin RNA levels (measured by transcription, amplification with the primers shown below, and quantification as described above). Hydrodynamic delivery of GFP-TMPRSS6 significantly reduced hepcidin RNA in the liver (Figure 6G). Treatment with 10 mg / kg of the MWTx-003 anti-TMPRSS6 antibody (Figures 6G-6H) or its humanized variant, hzMWTx-003Var anti-TMPRSS6 antibody (Figure 6I), reversed the suppression of Hamp and significantly increased hepcidin RNA levels in the liver. The following primers were used for RNA quantification by real-time qPCR: hepcidin forward primer: 5'-AAG CAG GGC AGA CAT TGC GAT-3' (SEQ ID NO: 85); hepcidin reverse primer: 5'-CAG GAT GTG GCT CTA GGC TAT-3' (SEQ ID NO: 86); β-actin forward primer: 5'-ACC CAC ACT GTG CCC ATC TA-3' (SEQ ID NO: 87); β-actin reverse primer: 5'-CAC GCT CGG TCA GGA TCT TC-3' (SEQ ID NO: 88).

[0161] Serum concentrations of the MWTx-003 anti-TMPRSS6 antibody or its humanized variant, hzMWTx-003Var anti-TMPRSS6 antibody, were quantified by an in-house developed cell surface ELISA (Figures 6J-6L, as described above). Briefly, diluted mouse serum or anti-TMPRSS6 antibody standards were incubated with 100% methanol-fixed HEK293T cells stably expressing human TMPRSS6 (HEK293T cells were used as a background control). Bound MWTx-003 anti-TMPRSS6 antibody was detected with goat anti-mouse IgG conjugated with HRP, and bound hzMWTx-003Var anti-TMPRSS6 antibody was detected with goat anti-human IgG conjugated with HRP. Color was developed with TMB followed by stop solution. Absorbance was then measured at OD . 450nm The results were read at 100 kHz. Samples were analyzed in triplicate, and results were normalized to HEK293T controls. Data were analyzed in Graphpad Prism 8 using a four-parameter logistic (4-PL) curve fit to interpolate serum anti-TMPRSS6 antibody concentrations.

[0162] [Example 7] In vivo efficacy of anti-TMPRSS6 antibodies using a beta-thalassemia mouse model. To test the in vivo efficacy of the anti-TMPRSS6 antibody, a β-thalassemia mouse model (B6.129P2-Hbb-b1) was used. tm1Unc Hbb-b2 tm1Unc Th3 / + mice (J, JAX Stock No: 002683, The Jackson Laboratories, Bar Harbor, ME) were selected and referred to here as Th3 / + mice. Four- to five-week-old Th3 / + mice and their wild-type (WT) littermates were fed an iron-replete diet (Teklad TD.80394). Th3 / + mice were treated with 10 mg / kg of MWTx-003 anti-TMPRSS6 antibody or mouse IgG2b isotype control every three days for four weeks, while WT littermates were untreated. After the treatment course, mice were euthanized, and spleens, livers, femurs, and blood samples were collected. Total liver RNA was purified, and serum was collected as described above.

[0163] Effects on blood counts, splenomegaly, serum iron, serum hepcidin, and liver hepcidin RNA Complete blood counts (CBCs) were performed on a VETSCAN HM5 automated hematology analyzer (Figures 7A-7D). Treatment with MWTx-003 anti-TMPRSS6 antibody significantly increased red blood cell counts (RBC, Figure 7A) and hematocrit (HCT, Figure 7C) and reduced red blood cell distribution width (RDW, Figure 7D) in Th3 / + mice, but had no apparent effect on hemoglobin (HGB, Figure 7B).

[0164] Measurement of spleen weight revealed that treatment with MWTx-003 anti-TMPRSS6 antibody significantly reduced splenomegaly in Th3 / + mice (Fig. 7E).

[0165] Serum iron was measured as described above. Treatment with MWTx-003 anti-TMPRSS6 antibody significantly reduced serum iron (Figure 7F). Liver non-heme iron was measured using a similar colorimetric assay (Figure 7G). Briefly, minced small liver tissue was dried overnight at 65°C and then digested with mixed acid (3 M HCl, 10% trichloroacetic acid) at 65°C for 20 hours. The digestion supernatant was then collected and developed with a color development solution (1.5 M sodium chloride, 0.5 mM bathophenanthroline disulfonate). The absorbance was then measured using an OD value. 535nm Treatment with MWTx-003 anti-TMPRSS6 antibody significantly reduced hepatic non-heme iron (Figure 7G).

[0166] Serum hepcidin was measured by the Hepcidin-Murine Compete ELISA kit as described above. Treatment with MWTx-003 anti-TMPRSS6 antibody significantly increased serum hepcidin (Figure 7H).

[0167] Liver hepcidin RNA was quantified by real-time qPCR as described above. Treatment with MWTx-003 anti-TMPRSS6 antibody significantly increased hepcidin RNA in the liver (Figure 7I).

[0168] Serum concentrations of MWTx-003 anti-TMPRSS6 antibody were quantified by an in-house developed cell surface ELISA as described above ( Figure 7J ).

[0169] Effects on erythropoiesis To test the effect of the MWTx-003 anti-TMPRSS6 antibody on erythropoiesis in Th3 / + mice, bone marrow was harvested from the femur (see Figures 7K-7M), and splenocytes were harvested from the spleen (Figures 7N-7P) and analyzed. Harvested cells were blocked with rat anti-mouse CD16 / CD32 (BD Biosciences) for 15 minutes, then stained with FITC-conjugated rat anti-mouse TER119 (BD Biosciences) and APC-conjugated rat anti-mouse CD44 (Invitrogen) for 30 minutes on ice. Washed cells were stained with the viability marker 7-AAD (BD Biosciences) for 10 minutes on ice before FACS analysis using a NOVOCYTE® flow cytometer. Ter119 + , 7-ADD -Cells were selected and cell size (FSC-H) was analyzed by anti-mouse CD44 antibody to generate density plots. The plots were analyzed to identify cell types (cell clusters) and determine the abundance of each cell type (cluster). According to Figures 7K-7P, four distinct cell clusters were identified from top to bottom, corresponding to successive stages of erythroid differentiation: basophilic erythroblasts (cluster I), polychromatic erythroblasts (cluster II), normochromatic erythroblasts and anucleated reticulocytes (cluster III), and mature erythrocytes (cluster IV). The percentage (%) value of each cluster in the sample was calculated as a measure of the abundance of that cell type in that cluster, as shown in Figures 7K-7P. For each sample (bone marrow, spleen) of each animal during each treatment course, the percentage values ​​of each cell cluster (I), (II), (III), and (IV) were calculated as follows: WT (untreated), N=9; disease model Th3 / + mice treated with IgG2b isotype control (Th3+ w / MoIgG2b), N=5; disease model Th3 / + mice treated with MWTx-003 anti-TMPRSS6 antibody (Th3+ w / MWTx-003), N=7, and the average values ​​were then calculated. On average, after 4 weeks, the basophilic erythroblast (I) population shifted from 7.58% (Th3+ w / MoIgG2b) to 6.52% (Th3+ w / MWTx-003) (7.96% in WT), the polychromatic erythroblast (II) population shifted from 54.20% (Th3+ w / MoIgG2b) to 40.01% (Th3+ w / MWTx-003) (28.53% in WT), the normochromatic erythroblast and non-nucleated reticulocyte (III) population shifted from 24.06% (Th3+ w / MoIgG2b) to 29.73% (Th3+ w / MWTx-003) (26.67% in WT), and the mature erythrocyte (IV) population shifted from 4.54% (Th3+ w / MoIgG2b) to 16.44% (27.66% in WT).On average, in the spleen after 4 weeks, the population of basophilic erythroblasts (I) showed a shift from 0.71% (Th3+ w / MoIgG2b) to 0.91% (Th3+ w / MWTx-003) (46% in WT), polychromatic erythroblasts (II) showed a shift from 45.76% (Th3+ w / MoIgG2b) to 19.25% (Th3+ w / MWTx-003) (12.23% in WT), normochromatic erythroblasts and anucleated reticulocytes (III) showed a shift from 31.16% (Th3+ w / MoIgG2b) to 28.72% (Th3+ w / MWTx-003) (8.67% in WT), and mature erythrocytes (IV) showed a shift from 14.13% (Th3+ w / MoIgG2b) to 44.38% (Th3+ The results are shown in bar graphs in Figure 7Q for bone marrow and in Figure 7R for spleen.

[0170] In Th3 / + mice, treatment with the MWTx-003 anti-TMPRSS6 antibody induced a significant proportion of erythroblasts to differentiate and mature into red blood cells, ameliorating inefficient erythropoiesis.

[0171] [Example 8] Epitope binning of anti-TMPRSS6 antibodies OCTET® RED96e was used for epitope binning of the MWTx-001 (Figure 8A), MWTx-002 (Figure 8B), and MWTx-003 (Figure 8C) anti-TMPRSS6 antibodies. First, ecto-TMPRSS6-FLAG (as described above) was labeled with biotin using a Biotinylation Kit (Abcam). Prehydrated streptavidin (SA) biosensors were equilibrated with 1x KB (as described above) for 60 seconds to establish a first baseline, after which 10 mg / ml of biotinylated ecto-TMPRSS6-FLAG was loaded onto the SA biosensor for 300 seconds. A second baseline signal was then established for 60 seconds before saturation with 50 mg / ml of antibody (MWTx-001, Figure 8A; MWTx-002, Figure 8B; MWTx-003, Figure 8C) in 1x KB for 600 seconds. Finally, a third baseline signal was established for 60 seconds, after which 50 μg / ml of MWTx-001, MWTx-002, or MWTx-003 was added in 1x KB for 300 seconds. The binding of the MWTx-001 anti-TMPRSS6 antibody to ecto-TMPRSS6-FLAG was not competed with that of the MWTx-002 or MWTx-003 anti-TMPRSS6 antibodies (Figure 8A). The binding of the MWTx-002 anti-TMPRSS6 antibody to ecto-TMPRSS6-FLAG was not competed with that of the MWTx-001 anti-TMPRSS6 antibody, but was competed with that of the MWTx-003 anti-TMPRSS6 antibody (Figure 8B). The binding of the MWTx-003 anti-TMPRSS6 antibody to ecto-TMPRSS6-FLAG did not compete with that of the MWTx-001 anti-TMPRSS6 antibody, but did compete with that of the MWTx-002 anti-TMPRSS6 antibody (Figure 8C). Data analysis was performed using Octet Data Analysis HT Software. The relevant signals are summarized in Figure 8D.

[0172] [Example 9] Efficacy test of anti-TMPRSS6 monoclonal antibody in a mouse model of polycythemia vera The effect of anti-TMPRSS6 recombinant monoclonal antibody treatment on reversing erythrocytosis and normalizing hematocrit levels in a mouse model of polycythemia vera (PV) was evaluated.

[0173] B6N.129S6(SJL)-Jak2 tm1.1Ble / AmlyJ mouse (JAX number 031658), commonly known as Jak2 V617F-Fl / + , is a floxed strain that harbors an inverted V617F mutation that places exon 14 downstream of the endogenous exon 14 of the Janus kinase 2 (Jak2) gene. The V617F mutation is common in patients with myeloproliferative neoplasms and is present in approximately 95% of PV patients. When crossed with mice expressing tissue-specific Cre recombinase, the resulting offspring have the floxed endogenous exon 14 removed and the V617F mutant exon 14 placed in the correct transcriptional orientation.

[0174] B6.Cg-Commd10 Tg(Vav1-icre)A2Kio The Jak2 / J mice (JAX number 008610), commonly known as Vav-iCre, express an optimized variant of Cre recombinase (iCre) specifically in hematopoietic cells and are useful for generating conditional mutations in the hematopoietic progenitor compartment. V617F-Fl / + Offspring of mice crossed with Vav-iCre transgenic mice develop PV, characterized by erythrocytosis and elevated hematocrit levels, and this phenotype can be propagated by transplanting bone marrow cells from the double transgenic mice into lethally irradiated wild-type recipient mice.

[0175] The recombinant mouse anti-TMPRSS6 monoclonal antibody MWTx-003, designated r4K12B in this study, is a recombinantly expressed version of the mouse monoclonal antibody MWTx-003 and can be referred to as recombinant monoclonal antibody MWTx-003, recombinant MWTx-003, or MWT-003, as shown in Figures 9A-9H. The recombinant mouse anti-TMPRSS6 monoclonal antibody r4K12B, the mouse version of the humanized antibody hzMWTx-003Var, was used in this in vivo repeated-dose study using the PV mouse model to avoid potential immunogenicity and the generation of anti-drug antibodies (ADAs). The recombinant mouse anti-TMPRSS6 monoclonal antibody r4K12B has an HC of SEQ ID NO: 69 (HC amino acid sequence of mouse monoclonal MWTx-003) and an LC of SEQ ID NO: 71 (LC amino acid sequence of mouse monoclonal MWTx-003) expressed from a vector into which the nucleotides of SEQ ID NO: 70 (HC coding sequence of MWTx-003) and SEQ ID NO: 72 (LC coding sequence of MWTx-003) were inserted by engineering an IRES between the HC coding sequence and the LC coding sequence, and the expressed polypeptide was purified.

[0176] material The following materials were used to evaluate the effect of anti-TMPRSS6 antibody treatment on reversing erythrocytosis and normalizing hematocrit levels in a mouse model of polycythemia vera. r4K12B, recombinant mouse monoclonal antibody (recombinant MWTx-003), homemade a. Isotype: Mouse IgG2b, kappa. b.Lot: LN211201 c. Concentration: 3.8mg / mL in PBS, pH 7.4 d. Purity: >95%, as determined by SDS-PAGE e. Endotoxin: 0.71EU / mg InVivoPlus Mouse IgG2b Isotype Control, purchased from BioXCell (number BP0086) f. Clone: ​​MPC-11 g.Lot:779420O1 h. Concentration: 10.26mg / mL in PBS, pH 7.0 i. Purity: >95% as determined by SDS-PAGE j. Endotoxin: Less than 1 EU / mg

[0177] method Animal experiments Wild-type C57BL / 6J (JAX No. 000664) male mice aged 10–12 weeks were purchased from Jackson Laboratory and acclimated to the breeding environment before the start of the study. All mice were subjected to lethal whole-body irradiation at a dose of 1000 cGy at 3.45 Gy / min. 24 hours later, Jak2 V617 / + 5 × 10 bone marrow cells isolated from Vav-iCre double transgenic mice (both males and females) 6 Each mouse was injected via the lateral tail vein into lethally irradiated recipient C57BL / 6J mice. Immediately after bone marrow transplantation (BMT), the mice were given free access to antibiotics (sulfamethoxazole and trimethoprim) in acidified drinking water (pH 2.5–3.0) for two weeks. BMT animals were monitored for the development of the PV phenotype by complete blood count using an automated hematology analyzer. Four weeks after BMT, when the PV phenotype was fully established, the mice were intraperitoneally injected with the anti-TMPRSS6 antibody r4K12B (recombinant MWTx-003) or a mouse IgG2b isotype control antibody once every four days for a total of three weeks. Four days after the final injection, the animals were euthanized, and bone marrow, spleen, liver, and whole blood were collected and analyzed. The effects of anti-TMPRSS6 antibody treatment on mouse erythrocyte profiles, hematological parameters (including mean corpuscular volume (MCV) and mean red blood cell size), splenomegaly, and tissue iron deposition were evaluated.

[0178] Serum hepcidin, iron concentration and tissue iron deposition

[0179] Serum hepcidin was measured by Hepcidin-Murine Compete™ ELISA (Intrinsic Lifesciences, SKU number HMC-001) according to the manufacturer's instructions as described above. The results are shown in (Figure 9E).

[0180] Serum iron was measured using a chromogenic assay as described above.

[0181] Iron deposition in the spleen and liver was assessed by Perls' Prussian blue staining of 10% formalin-fixed liver and spleen sections. Sectioning, staining, and imaging were performed by Reveal Biosciences (San Diego, California) (Figure 9H).

[0182] Analysis of hematological parameters Red blood cell indices were analyzed by complete blood count (CBC) on an HM5 VetScan Hematology Analyzer (Figures 9A-9C).

[0183] Erythroblast differentiation was assessed in bone marrow and spleen, respectively. Bone marrow harvested from femurs and splenocytes harvested from spleens were analyzed by FACS as described above. The results are shown in Figure 9G.

[0184] Measurement of anti-TMPRSS6 antibody concentrations in mouse serum Serum concentrations of r4K12B anti-TMPRSS6 antibody (recombinant MWTx-003) were quantified by a cell surface ELISA developed in-house as described above, and the results are shown in (Figure 9F).

[0185] statistical analysis One-way analysis of variance was used to compare three or more data sets using GraphPad Prism software. P<0.05 was considered statistically significant.

[0186] result Animal Grouping The body weights of bone marrow recipient C57BL / 6J mice (all males) were measured during acclimation for randomization to ensure comparable mean body weights among groups. Grouping was performed according to the following table (Table 4).

[0187] [Table 4]

[0188] Jak2 V617 / + Expression of PV phenotype in mice injected with Vav-iCre bone marrow cells

[0189] Blood samples were collected from recipient mice (wild-type C57BL / 6J mice that underwent bone marrow transplantation (BMT) of Jak2V617 / + Vav-iCre bone marrow cells) and hematological parameters were analyzed 3 and 4 weeks after BMT, respectively. V617 / + With reference to the Vav-iCre double transgenic mice (referred to as the “PV reference line”), the PV phenotype developed in recipient mice 3 weeks after BMT and was fully established 4 weeks after BMT (Table 5 ).

[0190] [Table 5]

[0191] Jak2 V617 / + Administration of anti-TMPRSS6 antibody reversed erythrocytosis and normalized hematocrit levels in a mouse PV model. Four weeks after BMT, when the PV phenotype was fully established, mice (referred to as "PV phenotype" mice) were intraperitoneally injected with the anti-TMPRSS6 antibody r4K12B (recombinant MWTx-003) at dose levels of 2 mg / kg, 5 mg / kg, or 10 mg / kg, or with a mouse IgG2b isotype control at 10 mg / kg every four days for three weeks. Endpoint analyses were performed four days after the final injection.

[0192] After 2 weeks of treatment with the anti-TMPRSS6 antibody r4K12B, a dose-dependent trend toward decreased hematocrit (HCT) levels, red blood cell (RBC) counts, and hemoglobin (HGB) concentrations was observed in mice treated with r4K12B compared with animals treated with an isotype control antibody (Table 6).

[0193] [Table 6]

[0194] Results 3 weeks after treatment Figures 9A-9C show the endpoint measurements of hematological parameters HCT (Figure 9A), RBC (Figure 9B), and HGB (Figure 9C) for each treatment and dose level. Figures 9D-9E also show the endpoint measurements for each treatment and dose level: Figure 9D shows splenomegaly (splenomegaly index measured as mg / g body weight), Figure 9E shows serum hepcidin levels (ng / ml), and Figure 9F shows serum anti-TMPRSS6 r4K12B concentrations (μg / ml) measured by cell surface ELISA. Figure 9G shows FACS results for measuring early erythroid precursors (cluster I, basophilic erythroblasts and cluster II, polychromatic erythroblasts) in the bone marrow (top row) and spleen (bottom row) in WT (left panel, top and bottom rows), MoIgG2b isotype control (middle panel, top and bottom rows), and 10 mg / kg anti-TMPRSS6 r4K12B (MWTx-003) treatment (right panel, top and bottom rows). Figure 9H shows sections of the liver (left panel) and spleen (right panel) stained for iron content. In Figures 9A-9H, the MWTx-003 label indicates treatment or measurement with antibody r4K12B.

[0195] At the end of the 3-week treatment period, HCT levels in all r4k12B-treated groups further decreased in a dose-dependent manner, reaching levels similar to or below those seen in wild-type (WT) untreated animals (Figure 9A). Circulating red blood cell counts and HGB concentrations also decreased, with a significant reduction in erythrocytosis noted in the 10 mg / kg treatment group (Figure 9B-C). Splenomegaly (Figure 9D) and expansion of early erythroid progenitors, i.e., cluster I, basophilic erythroblasts, and cluster II, polychromatic erythroblasts (Figure 9G), were also observed in the 10 mg / kg treatment group, indicating the onset of iron-restricted erythropoiesis. As expected, serum hepcidin levels significantly increased and persisted throughout the treatment period (Figure 9E), resulting in a dramatic decrease in serum iron concentrations below the detection limit by colorimetric assay (data not shown). These observations suggest that the anti-TMPRSS6 antibody r4K12B (MWTx-003) is effective in suppressing erythrocytosis and ameliorating the PV phenotype, but the dose and duration of treatment should be gradually increased to minimize the adverse effects of erythrocytic iron deficiency. Figure 9G shows representative FACS results measuring early erythroid precursors in the bone marrow (top row) and spleen (bottom row). Cluster I represents basophilic erythroblasts, while Cluster II represents polychromatic erythroblasts. The results are shown for WT (left panel, top and bottom rows), MoIgG2b isotype control (middle panel, top and bottom rows), and 10 mg / kg anti-TMPRSS6 r4K12B (MWTx-003) treatment (right panel, top and bottom rows). The combined percentage of cluster I and II erythroid progenitors in the spleen was 22.17 ± 1.74, 24.09 ± 4.52, and 40.06 ± 10.04 in the wild-type control, 10 mg / kg moIgG2b, and 10 mg / kg r4K12B groups, respectively. The combined percentage of cluster I and II in the r4K12B group was statistically different from that in the moIgG2b and wild-type control mice (P = 0.0399 and P = 0.0277, respectively), but there was no statistical difference between the wild-type and moIgG2b-treated groups.

[0196] Figure 9H shows Perls' Prussian blue staining of formalin-fixed liver (left panel) and spleen (right panel) sections from control animals treated with the mouse IgG2b isotype control MoIgG2b (top row) and animals treated with increasing doses of anti-TMPRSS6 r4K12B (labeled MWTx-003) as indicated to measure iron deposition. The results in Figure 9H show that administration of anti-TMPRSS6 antibody r4K12B (MWTx-003) did not significantly alter liver iron content but did cause a significant increase in iron deposition in splenic macrophages, and the increase was observed in a dose-dependent manner.

[0197] conclusion Subchronic treatment with anti-TMPRSS6 antibody significantly reduced erythrocytosis and normalized hematocrit levels in a mouse model of polycythemia vera by restricting iron availability to erythroid precursors. Anti-TMPRSS6 antibody therapy represents a promising therapeutic approach for the management of PV, in which erythrocytosis and high HCT levels are associated with poor prognosis.

Claims

1. 1. A method for treating polycythemia vera (PV) in a subject, wherein the PV is associated with overactivation of the JAK2 / STAT5 pathway, the method comprising administering to the subject an effective amount of an anti-TMPRSS6 antibody, wherein the antibody: (a) a heavy chain complementarity determining region 1 (HC CDR1) comprising the amino acid sequence of SEQ ID NO:52, a HC CDR2 comprising the amino acid sequence of SEQ ID NO:53, a HC CDR3 comprising the amino acid sequence of SEQ ID NO:54, a light chain complementarity determining region 1 (LC CDR1) comprising the amino acid sequence of SEQ ID NO:57, a LC CDR2 comprising the amino acid sequence of SEQ ID NO:58, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO:59; (b) a HC CDR1 comprising the amino acid sequence of SEQ ID NO:2, a HC CDR2 comprising the amino acid sequence of SEQ ID NO:3, a HC CDR3 comprising the amino acid sequence of SEQ ID NO:4, a LC CDR1 comprising the amino acid sequence of SEQ ID NO:7, a LC CDR2 comprising the amino acid sequence of SEQ ID NO:8, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO:9; (c) a HC CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a HC CDR2 comprising the amino acid sequence of SEQ ID NO: 13, a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 14, a LC CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO: 19; (d) a HC CDR1 comprising the amino acid sequence of SEQ ID NO:22, a HC CDR2 comprising the amino acid sequence of SEQ ID NO:23, a HC CDR3 comprising the amino acid sequence of SEQ ID NO:24, a LC CDR1 comprising the amino acid sequence of SEQ ID NO:27, a LC CDR2 comprising the amino acid sequence of SEQ ID NO:28, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO:29; (e) a HC CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a HC CDR2 comprising the amino acid sequence of SEQ ID NO: 33, a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 34, a LC CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO: 39; or (f) a HC CDR1 comprising the amino acid sequence of SEQ ID NO: 42, a HC CDR2 comprising the amino acid sequence of SEQ ID NO: 43, a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 44, a LC CDR1 comprising the amino acid sequence of SEQ ID NO: 47, a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 48, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO:

49. A method comprising:

2. 1. A method for treating polycythemia vera (PV) in a subject having bone marrow containing cells with JAK2 / STAT5 hyperactivation, comprising administering to the subject an effective amount of an anti-TMPRSS6 antibody, wherein the antibody: (a) a heavy chain complementarity determining region 1 (HC CDR1) comprising the amino acid sequence of SEQ ID NO:52, a HC CDR2 comprising the amino acid sequence of SEQ ID NO:53, a HC CDR3 comprising the amino acid sequence of SEQ ID NO:54, a light chain complementarity determining region 1 (LC CDR1) comprising the amino acid sequence of SEQ ID NO:57, a LC CDR2 comprising the amino acid sequence of SEQ ID NO:58, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO:59; (b) a HC CDR1 comprising the amino acid sequence of SEQ ID NO:2, a HC CDR2 comprising the amino acid sequence of SEQ ID NO:3, a HC CDR3 comprising the amino acid sequence of SEQ ID NO:4, a LC CDR1 comprising the amino acid sequence of SEQ ID NO:7, a LC CDR2 comprising the amino acid sequence of SEQ ID NO:8, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO:9; (c) a HC CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a HC CDR2 comprising the amino acid sequence of SEQ ID NO: 13, a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 14, a LC CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO: 19; (d) a HC CDR1 comprising the amino acid sequence of SEQ ID NO:22, a HC CDR2 comprising the amino acid sequence of SEQ ID NO:23, a HC CDR3 comprising the amino acid sequence of SEQ ID NO:24, a LC CDR1 comprising the amino acid sequence of SEQ ID NO:27, a LC CDR2 comprising the amino acid sequence of SEQ ID NO:28, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO:29; (e) a HC CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a HC CDR2 comprising the amino acid sequence of SEQ ID NO: 33, a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 34, a LC CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO: 39; or (f) a HC CDR1 comprising the amino acid sequence of SEQ ID NO: 42, a HC CDR2 comprising the amino acid sequence of SEQ ID NO: 43, a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 44, a LC CDR1 comprising the amino acid sequence of SEQ ID NO: 47, a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 48, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO:

49. A method comprising:

3. 3. The method of claim 1 or 2, wherein the subject has a mutation that leads to overactivation of JAK2 / STAT5.

4. The method of any one of claims 1 to 3, wherein the subject has a JAK2 mutation.

5. The method of claim 4, wherein the JAK2 mutation is a JAK2 gene exon 14 mutation.

6. 6. The method of claim 5, wherein the JAK2 exon 14 mutation is V617F, H606Q, H608Y, L611V, L611S, V617I, C618F, C618R, or an absence of exon 14.

7. 7. The method of claim 5 or 6, wherein the JAK2 exon 14 mutation is V617F.

8. 8. The method of claim 7, wherein the subject is homozygous for the JAK2 V617F mutation.

9. The method according to any one of claims 1 to 8, wherein the JAK2 mutation is a mutation in exon 12 of the JAK2 gene.

10. The JAK2 mutations are F537-K539delinsL, N542-E543del mutations, H538QK539L, V536-I546 dup11, V536-F547 10. The method of claim 9, wherein the nucleotide sequence is selected from the group consisting of dup, F537-I546dup10F547L, F537IK539I, H538-K539delinsL, H538-K539del, H538DK539LI540S, H538G, K539L, K539E, I540-E543delinsMK, I540-E542delinsS, R541-E543delinsK, N542-E543del, D544-L545del, or 547insLI540-F547dup8.

11. The method according to any one of claims 1 to 10, wherein the subject has a mutation in exon 15 of the JAK2 gene.

12. The method of claim 11, wherein the JAK2 gene exon 15 mutation is L642P or I645V.

13. The method of any one of claims 1 to 12, wherein the subject has a non-Jak2 mutation.

14. The subject is a patient having a gene encoding the SRSF2 gene, the SF3B1 gene, the U2AF1 gene, the U2AF1 gene, the ZRSR2 gene, the TET2 gene, the DNMT3a gene, the IDH1 / IDH2 gene, the ASXL1 gene, the EZH2 gene, the LNK / SH2B3 gene, the NF-E2 gene, the NF1 gene, the CBL gene, the FLT3 gene, the ERBB gene, the PPM1D gene, the TR53 gene, the RUNX1 gene, the CUX1 gene, the ETV6 gene, the CALR gene 14. The method of claim 13, wherein the patient has a mutation in the MPL gene, let-7a gene, miR-26b gene, miR-27b gene, miR-28 gene, miR-30b gene, miR-30c gene, miR-125-5p gene, miR-125b-5p gene, miR-143 gene, miR-145 gene, miR-150 gene, miR-182 gene, miR-223 gene, miR-342 gene, or miR-451 gene.

15. The method according to any one of claims 3 to 14, wherein the mutation is an acquired mutation, a familial mutation, or a congenital mutation.

16. The method of any one of claims 1 to 15, wherein the subject comprises hematopoietic progenitor cells containing one or more of the mutations.

17. The method of claim 16, wherein the one or more mutations occur in CD34+CD38- hematopoietic progenitor cells.

18. 18. The method of claim 16 or 17, wherein the one or more mutations occur in a myeloid progenitor cell.

19. The method of any one of claims 16 to 18, wherein the one or more mutations occur in megakaryocyte-erythroid progenitor cells.

20. 20. The method of any one of claims 1 to 19, wherein the subject exhibits a PV phenotypic profile prior to said administration.

21. 21. The method of any one of claims 1 to 20, wherein the subject has an increased hematocrit (HCT) compared to a subject without PV.

22. The method of any one of claims 1 to 21, wherein the subject has splenomegaly prior to said administering.

23. 23. The method of any one of claims 1 to 22, wherein the subject has polycythemia prior to said administering.

24. The method of any one of claims 1 to 23, wherein the subject has leukocytosis prior to said administering.

25. The method of any one of claims 1 to 24, wherein the subject has thrombocytosis prior to said administering.

26. 26. The method of any one of claims 1 to 25, wherein the subject has increased hemoglobin compared to a subject without PV prior to said administration.

27. 27. The method of any one of claims 1 to 26, wherein the subject has an increased red blood cell distribution width (RDW) compared to a subject without PV prior to said administration.

28. 28. The method of any one of claims 1 to 27, wherein administration of the antibody increases serum hepcidin.

29. 29. The method of any one of claims 1 to 28, wherein administration of the antibody reduces liver iron.

30. 30. The method of any one of claims 1 to 29, wherein administration of the antibody reduces HCT.

31. 31. The method of any one of claims 1 to 30, wherein administration of the antibody reduces red blood cell count.

32. 32. The method of any one of claims 1 to 31, wherein administration of the antibody reduces red blood cell distribution width (RDW).

33. 33. The method of any one of claims 1 to 32, wherein administration of the antibody reduces serum iron.

34. 34. The method of any one of claims 1 to 33, wherein administration of the antibody reduces leukocytosis.

35. 35. The method of any one of claims 1 to 34, wherein administration of the antibody reduces early erythroid progenitors.

36. 36. The method of any one of claims 1 to 35, wherein administration of the antibody reduces plasma hemoglobin levels.

37. 36. The method of any one of claims 1 to 35, wherein administration of the antibody reduces mean corpuscular volume (MCV).

38. 38. The method of any one of claims 1 to 37, wherein administration of the antibody reduces the frequency of thrombotic events (TE).

39. 39. The method of any one of claims 1 to 38, wherein administration of the antibody reduces the frequency of phlebotomy.

40. 40. The method of any one of claims 1 to 39, wherein administration of the antibody reduces the frequency of cytoreductive therapy.

41. The method of any one of claims 1 to 40, wherein the antibody treats a subject in need thereof who is refractory to phlebotomy and / or cytoreductive therapy.

42. 42. The method of any one of claims 1-41, wherein administration of the antibody results in a reduction in symptoms as documented on the Myeloproliferative Neoplasm Symptom Assessment Form (MPN-SAF).

43. 43. The method of any one of claims 1-42, wherein the subject receives one or more additional therapeutic agents for treating PV.

44. Additional therapeutic agents for treating PV include interferons [e.g., lopeginterferon a-2b-njft (Besremi), pegylated interferon], JAK2 inhibitors (e.g., ruxolitinib, XL019, fedratinib (SAR302503), momelotinib), JAK1 inhibitors (e.g., itacitinib), hepcidin memory agents [e.g., rusfeltide (PTG-300)], lysine-specific demethylase inhibitors [e.g., bomedemstat (IMG-7298)], TMPRSS6 antagonists [e.g., sapablursen (ISIS 702843), SLN1 24], an anti-TfR1 antibody (e.g., PPMX-T003), an MDM2 inhibitor [e.g., Idasanutlin (RG7388), KRT-232], a tyrosine kinase inhibitor [e.g., Dasatinib, Erlotinib, Gleevec, lestaurtinib (CEP-701)], an HDAC inhibitor [e.g., Givinostat (ITF2357), MK-0683], a PI3K inhibitor [e.g., Umbralisib (TGR-1202)], a telomerase inhibitor (e.g., Imetelstat), phlebotomy, low-dose aspirin, or hydroxyurea.

43. 43. The method of any one of claims 1 to 42, wherein the antibody comprises a heavy chain complementarity determining region 1 (HC CDR1) comprising the amino acid sequence of SEQ ID NO: 52, a HC CDR2 comprising the amino acid sequence of SEQ ID NO: 53, a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 54, a light chain complementarity determining region 1 (LC CDR1) comprising the amino acid sequence of SEQ ID NO: 57, a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 58, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO:

59.

44. The antibody (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 51, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 56; (b) a VH comprising the amino acid sequence of SEQ ID NO: 1, and a VL comprising the amino acid sequence of SEQ ID NO: 6; (c) a VH comprising the amino acid sequence of SEQ ID NO: 11, and a VL comprising the amino acid sequence of SEQ ID NO: 16; (d) a VH comprising the amino acid sequence of SEQ ID NO: 21, and a VL comprising the amino acid sequence of SEQ ID NO: 26; (e) a VH comprising the amino acid sequence of SEQ ID NO: 31, and a VL comprising the amino acid sequence of SEQ ID NO: 36; or (f) a VH comprising the amino acid sequence of SEQ ID NO: 41, and a VL comprising the amino acid sequence of SEQ ID NO: 46; 44. The method of any one of claims 1 to 43, comprising:

45. 45. The method of any one of claims 1 to 44, wherein the antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 51, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:

56.

46. The antibody (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 81, and a light chain comprising the amino acid sequence of SEQ ID NO: 83; (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 61, and a light chain comprising the amino acid sequence of SEQ ID NO: 63; (c) a heavy chain comprising the amino acid sequence of SEQ ID NO: 65, and a light chain comprising the amino acid sequence of SEQ ID NO: 67; (d) a heavy chain comprising the amino acid sequence of SEQ ID NO: 69, and a light chain comprising the amino acid sequence of SEQ ID NO: 71; (e) a heavy chain comprising the amino acid sequence of SEQ ID NO: 73, and a light chain comprising the amino acid sequence of SEQ ID NO: 75; or (f) a heavy chain comprising the amino acid sequence of SEQ ID NO: 77, and a light chain comprising the amino acid sequence of SEQ ID NO:

79.

46. ​​The method of any one of claims 1 to 45, comprising:

47. The method of any one of claims 1 to 46, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 81 and a light chain comprising the amino acid sequence of SEQ ID NO:

83.

48. 48. The method of any one of claims 1 to 47, wherein the antibody comprises a heavy chain complementarity determining region 1 (HC CDR1) comprising the amino acid sequence of SEQ ID NO: 52, a HC CDR2 comprising the amino acid sequence of SEQ ID NO: 53, a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 54, a light chain complementarity determining region 1 (LC CDR1) comprising the amino acid sequence of SEQ ID NO: 57, a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 58, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO:

59.

49. The antibody (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 51, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 56; (b) a VH comprising the amino acid sequence of SEQ ID NO: 1, and a VL comprising the amino acid sequence of SEQ ID NO: 6; (c) a VH comprising the amino acid sequence of SEQ ID NO: 11, and a VL comprising the amino acid sequence of SEQ ID NO: 16; (d) a VH comprising the amino acid sequence of SEQ ID NO: 21, and a VL comprising the amino acid sequence of SEQ ID NO: 26; (e) a VH comprising the amino acid sequence of SEQ ID NO: 31, and a VL comprising the amino acid sequence of SEQ ID NO: 36; or (f) a VH comprising the amino acid sequence of SEQ ID NO: 41, and a VL comprising the amino acid sequence of SEQ ID NO: 46; 49. The method of any one of claims 1 to 48, comprising:

50. 50. The method of any one of claims 1 to 49, wherein the antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 51, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:

56.

51. The antibody (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 81, and a light chain comprising the amino acid sequence of SEQ ID NO: 83; (b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 61, and a light chain comprising the amino acid sequence of SEQ ID NO: 63; (c) a heavy chain comprising the amino acid sequence of SEQ ID NO: 65, and a light chain comprising the amino acid sequence of SEQ ID NO: 67; (d) a heavy chain comprising the amino acid sequence of SEQ ID NO: 69, and a light chain comprising the amino acid sequence of SEQ ID NO: 71; (e) a heavy chain comprising the amino acid sequence of SEQ ID NO: 73, and a light chain comprising the amino acid sequence of SEQ ID NO: 75; or (f) a heavy chain comprising the amino acid sequence of SEQ ID NO: 77, and a light chain comprising the amino acid sequence of SEQ ID NO:

79.

51. The method of any one of claims 1 to 50, comprising:

51. The method of any one of claims 1 to 50, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 81 and a light chain comprising the amino acid sequence of SEQ ID NO:

83.

52. An anti-TMPRSS6 antibody according to any one of claims 1 to 52, wherein the antibody cross-reacts with at least one non-human TMPRSS6.

53. The anti-TMPRSS6 antibody of claim 52, wherein the non-human TMPRSS6 is mouse TMPRSS6 or non-human primate TMPRSS6.

54. An anti-TMPRSS6 antibody according to any one of claims 1 to 53, wherein the antibody specifically binds to human TMPRSS6.

55. The anti-TMPRSS6 antibody of any one of claims 1 to 54, wherein the antibody does not specifically bind to human matriptase-1 or human matriptase-3.

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