Anti-TMPRSS6 antibodies and uses thereof

Abstract

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

Description

Field of the Invention

[0001] The present disclosure relates to antibodies and antigen-binding fragments that bind TMPRSS6, and the use of antibodies and antigen-binding fragments that bind TMPRSS6 to treat disorders including iron metabolism disorders and myeloproliferative neoplasms. Background of the Invention

[0003] Type II transmembrane serine protease 6 (TMPRSS6) is encoded by the TMPRSS6 gene and is primarily expressed in the liver. The structure of TMPRSS6 includes a type II transmembrane domain, followed by sea urchin sperm protein, enteropeptidase, and agrin (SEA) domains, a stem region containing two complement factor C1r / C1s, sea urchin embryonic growth factor, and bone morphogenetic protein (CUB) domains, and 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 serine protease 6; membrane-bound mosaic serine protease matriptase-2; and MT2.

[0004] TMPRSS6 plays an important role in iron homeostasis through the BMP-SMAD signaling pathway that regulates the expression of hepcidin, a hormone that controls iron absorption and mobilization from iron reserves. Hepcidin (also known as: HAMP (hepcidin antimicrobial protein or peptide), encoded by HAMP in humans and non-human primates, and encoded by Hamp in mice and rats) regulates systemic iron homeostasis by controlling the functional activity of the only iron efflux channel, ferroportin. Hepcidin can reduce plasma iron levels by binding to ferroportin and causing internalization and degradation of the complex, thereby preventing iron absorption and release of stored iron in the small intestine. Chronic elevation of hepcidin levels causes systemic iron deficiency, while hepcidin deficiency causes systemic iron overload.

[0005] TMPRSS6 negatively regulates hepcidin production through a transmembrane signaling pathway that is triggered by iron deficiency and suppresses 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 trigger this pathway to reduce hepcidin production, which allows more iron from the diet to be absorbed through the intestine and transported from storage sites into the bloodstream. In rats under acute iron deprivation, liver TMPRSS6 protein levels are upregulated, leading to suppressed hepcidin expression and production (Wang, C.-Y. et al., Front. Pharmacol. . 2014.5:114). Mutations throughout the TMPRSS6 molecule, and particularly in the extracellular domain, have been identified in subjects with iron deficiency anemia, particularly iron-refractory iron deficiency anemia (IRIDA), which is unresponsive to oral iron therapy and only partially responsive to parenteral iron therapy (Wang, C.-Y. et al., Front. Pharmacol. . Loss-of-function mutations in TMPRSS6 in humans lead to elevated hepcidin levels and iron-deficiency anemia (Camaschella, C., N Engl Journal Med 2013. 168: 24), as overproduction of hepcidin leads to defective iron absorption and utilization.

[0006] When excess iron accumulates in tissues and organs to the extent that their normal functions are disrupted, an iron overload disorder results. Iron toxicity is a common complication of iron overload disorders, resulting in a high mortality rate due to iron accumulation in major organs. β-thalassemia is an iron overload disorder that occurs when a mutation in the HBB gene causes a reduction in or absence of the production of β-globin (β-globin), which results in apoptosis of nucleated red blood cells and a deficiency of mature red blood cells, resulting in ineffective erythropoiesis, which causes anemia and excessive absorption of iron, leading to iron poisoning. In patients with β-thalassemia, hepcidin is abnormally suppressed compared to the patient's iron load status, causing hepcidin deficiency, which in turn allows excessive iron absorption and the development of systemic iron overload. Other disorders such as MDS (myelodysplastic syndrome), dyserythropoietic anemia, and ineffective erythropoiesis in sideroblastic anemia are similarly characterized by low hepcidin, resulting in iron overload. Hemochromatosis, such as type 1 hemochromatosis or hereditary hemochromatosis, is a kind of iron overload disease, it is characterized in that the excessive intestinal absorption of dietary iron and the pathological increase of systemic iron reserves.The current standard of care for treating iron overload disease includes blood transfusion for invalid erythropoiesis, which can further aggravate iron overload, iron chelation with weak patient compliance, and phlebotomy or splenectomy for responding to symptoms.The therapeutic method currently under development includes gene therapy targeting HBB gene, gene therapy and gene editing targeting other related genes, hepcidin mimics, targeting TGF superfamily ligand to suppress the Fc fusion protein of SMAD signal conduction, antisense RNA drugs targeting TMPRSS6 (for example, El-Beshlawy A., et al., Blood Cells, Molecules and Diseases 2019.76:53-58) and iRNA drugs targeting TMPRSS6.

[0007] Polycythemia vera (PV) is a chronic myeloproliferative neoplasm with constitutively activated JAK2 / STAT5 signaling, leading to increased red blood cell clumps and erythroid hyperplasia. The primary cause of mortality is thrombotic complications due to high blood viscosity. Potential downstream effects of constitutively activated JAK2 / STAT5 signaling may include concurrent abnormal erythropoiesis, an inflammatory milieu, decreased systemic iron concentrations, and potentially altered hypoxic responsiveness, which may directly affect iron absorption in certain tissues. Any or all of these may play a role in iron metabolism in PV. (Ginzburg, YZ, et al., Leukemia 2018. 32:2105–2116) Evidence suggests that systemic iron deficiency or erythroid-targeted iron restriction may be beneficial in reducing polycythemia and normalizing hematocrit in PV. SUMMARY OF THE INVENTION

[0009] The present invention relates to novel antibodies and antigen-binding fragments thereof that bind to TMPRSS6, as well as methods of making and using antibodies and antigen-binding fragments thereof that bind to TMPRSS6.

[0010] The present disclosure provides anti-TMPRSS6 antibodies, nucleic acids encoding anti-TMPRSS6 antibodies, and methods for preparing and using anti-TMPRSS6 antibodies. Anti-TMPRSS6 antibodies as disclosed herein encompass anti-TMPRSS6 antibodies and fragments thereof that are capable of binding to TMPRSS6. Anti-TMPRSS6 antibodies as 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 purposes. Anti-TMPRSS6 antibodies as disclosed herein can be used to treat iron metabolism disorders, such as iron overload disorders, particularly β-thalassemia, including but not limited to non-transfusion-dependent thalassemia, and other disorders of ineffective erythropoiesis. Anti-TMPRSS6 antibodies as disclosed herein can be used to treat myeloproliferative disorders, such as polycythemia vera (PV), which is characterized by polycythemia vera and erythroid hyperplasia.

[0011] In one aspect, an anti-TMPRSS6 antibody is provided that is capable of binding to TMPRSS6 on the surface of a cell expressing TMPRSS6 and modulating the activity of at least one component involved in iron metabolism, wherein the component may be a molecule or biological process related to the function of TMPRSS6. In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein are capable of modulating the activity of at least one component involved in regulating hepcidin expression. In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein are capable of substantially inhibiting TMPRSS6 suppression of hepcidin expression. In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein are capable of increasing hepcidin expression. In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein are capable of increasing the activity of the hepcidin promoter. In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein are capable of substantially inhibiting TMPRSS6 suppression of hepcidin expression induced by the BMP / SMAD pathway. The anti-TMPRSS6 antibodies disclosed herein may modulate hepcidin expression in a dose-dependent manner, including but not limited to TMPRSS6 suppression that substantially inhibits hepcidin expression, increases hepcidin expression, increases hepcidin promoter activity, or TMPRSS6 suppression that substantially inhibits BMP / SMAD pathway-induced hepcidin expression. In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein are capable of modulating hepcidin expression in a dose-dependent manner. In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein are capable of increasing serum hepcidin levels in a dose-dependent manner when administered to a subject. In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein are capable of reducing serum iron levels in a dose-dependent manner when administered to a subject. In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein are capable of increasing liver hepcidin RNA levels in a dose-dependent manner when administered to a subject. In certain embodiments, when administered to a subject known or suspected of having an iron overload disorder, particularly β-thalassemia, the anti-TMPRSS6 antibodies disclosed herein are capable of 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 the production of mature red blood cells (increasing erythropoiesis). In certain embodiments, when administered to a subject known or suspected of having a myeloproliferative disorder, such as a myeloproliferative neoplasm, particularly polycythemia vera (PV), the anti-TMPRSS6 antibodies disclosed herein are capable of reducing RBC, reducing HCT, reducing hemoglobin (HGB), reducing mean corpuscular volume (MCV), and reducing RDW.

[0012] 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.

[0013] 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 show detectable binding to a matriptase homolog. In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein bind to human TMPRSS6 (matriptase-2) and do not show 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 show 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 show detectable binding to human matriptase-1 (ST14) or human matriptase-3 (TMPRSS7).

[0014] 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 (VHH, nanobodies), or other TMPRSS6 binding fragments or variants. In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein may comprise a framework in which amino acids have been substituted into an existing antibody framework, particularly to affect properties such as antigen binding ability. In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein may comprise complementarity determining regions (CDRs) from a source (parent) antibody that have been grafted (fused) into a framework from an antibody of a different type (class) than the parent antibody and / or a different organism, particularly an acceptor human framework. In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein may comprise a framework in which amino acids have been substituted, mutated, or replaced in regions outside of the CDRs to affect properties such as antigen binding or antibody structure, for example, in the variable region framework and / or constant region, particularly the Fc region, surrounding the CDRs. 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.

[0015] In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein comprise at least one polypeptide having an amino acid sequence as shown in Table 1, Table 2, or Table 3, or a sequence having substantial identity (e.g., at least 85%, 90%, 92%, 95%, 97%, or 98%, 99% identity) to an amino acid sequence as shown in Table 1, Table 2, or Table 3. The anti-TMPRSS6 antibodies disclosed herein may comprise at least one polypeptide having an amino acid sequence selected from the group consisting of 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: 35; SEQ ID NO: 36; SEQ ID NO: 37; SEQ ID NO: 38; SEQ ID NO: 39; SEQ ID NO: 40 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 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 NO:61; SEQ ID NO:63; SEQ ID NO:65; SEQ ID NO:67; SEQ ID NO:69; SEQ ID NO:71; ID NO:73; SEQ ID NO:75; SEQ ID NO:77; SEQ ID NO:79; SEQ ID NO:81; or SEQ ID NO:83.

[0016] In one embodiment, an anti-TMPRSS6 antibody disclosed herein comprises a heavy chain (HC) variable region polypeptide having an amino acid sequence as 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 an amino acid sequence as set forth in SEQ ID NO: 6, or a sequence substantially identical to SEQ ID NO: 6. In one embodiment, an anti-TMPRSS6 antibody disclosed herein comprises a heavy chain complementary determining region 1 (HC CDR1) having the amino acid sequence GYTFTSYW set forth in SEQ ID NO:2, a heavy chain complementary determining region 2 (HC CDR2) having the amino acid sequence IYPGSGST set forth in SEQ ID NO:3, a heavy chain complementary determining region 3 (HC CDR3) having the amino acid sequence APYDSDYAMDY set forth in SEQ ID NO:4; a light chain complementary determining region 1 (LC CDR1) having the amino acid sequence QDINNY set forth in SEQ ID NO:7, a light chain complementary determining region 2 (LC CDR2) having the amino acid sequence RAN set forth in SEQ ID NO:8, and a light chain complementary determining region 3 (LC CDR3) having the amino acid sequence LQYDEFPLT set forth in SEQ ID NO:9; or a variant of the antibody comprising 1, 2, 3, 4, 5 or 6 amino acid substitutions in the CDR regions. In one non-limiting embodiment, an 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.

[0017] In one embodiment, an anti-TMPRSS6 antibody disclosed herein comprises a 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 a 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 a HC CDR1 having the amino acid sequence GFNIKDYY set forth in SEQ ID NO: 12, a HC CDR2 having the amino acid sequence IDPEDGES set forth in SEQ ID NO: 13, a HC CDR3 having the amino acid sequence TRGDSMMVTYFDY set forth in SEQ ID NO: 14; a LC CDR1 having the amino acid sequence QDVSTA set forth in SEQ ID NO: 17, a LC CDR2 having the amino acid sequence WAF set forth in SEQ ID NO: 18, and a LC CDR3 having the amino acid sequence QQHYRSPWT set forth in SEQ ID NO: 19, or variants of such antibodies comprising 1, 2, 3, 4, 5, or 6 amino acid substitutions in the CDR regions. In one non-limiting embodiment, an anti-TMPRSS6 antibody disclosed herein is 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.

[0018] In one embodiment, an anti-TMPRSS6 antibody disclosed herein comprises a 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 a 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 a HC CDR1 having the amino acid sequence GFNIEDYY set forth in SEQ ID NO: 22, a HC CDR2 having the amino acid sequence IDPEDGET set forth in SEQ ID NO: 23, a HC CDR3 having the amino acid sequence ARSIYLDPMDY set forth in SEQ ID NO: 24; a LC CDR1 having the amino acid sequence QDVTTA set forth in SEQ ID NO: 27, a LC CDR2 having the amino acid sequence WAT set forth in SEQ ID NO: 28, and a LC CDR3 having the amino acid sequence QQHYSTPYT set forth in SEQ ID NO: 29, or variants of such antibodies comprising 1, 2, 3, 4, 5, or 6 amino acid substitutions in the CDR regions. In one non-limiting embodiment, an 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.

[0019] In one embodiment, an anti-TMPRSS6 antibody disclosed herein comprises a 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 a 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 a HC CDR1 having the amino acid sequence GYTFTSYW set forth in SEQ ID NO: 32, a HC CDR2 having the amino acid sequence IYPGSGST set forth in SEQ ID NO: 33, a HC CDR3 having the amino acid sequence APYDADYAMDY set forth in SEQ ID NO: 34; a LC CDR1 having the amino acid sequence QDISNY set forth in SEQ ID NO: 37, a LC CDR2 having the amino acid sequence RAN set forth in SEQ ID NO: 38, and a LC CDR3 having the amino acid sequence LQYDEFPLT set forth in SEQ ID NO: 39, or variants of such antibodies comprising 1, 2, 3, 4, 5, or 6 amino acid substitutions in the CDR regions. In one non-limiting embodiment, an anti-TMPRSS6 antibody disclosed herein is the antibody identified herein as the humanized anti-TMPRSS6 antibody variant hzMWTx-001Var, which comprises an HC polypeptide having the amino acid sequence shown in SEQ ID NO:73 and an LC polypeptide having the amino acid sequence shown in SEQ ID NO:75.

[0020] In one embodiment, an anti-TMPRSS6 antibody disclosed herein comprises a 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 a 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 a HC CDR1 having the amino acid sequence GFNIKDYY set forth in SEQ ID NO: 42, a HC CDR2 having the amino acid sequence IDPEDAES set forth in SEQ ID NO: 43, a HC CDR3 having the amino acid sequence TRGDSMMVTYFDY set forth in SEQ ID NO: 44; a LC CDR1 having the amino acid sequence QDVSTA set forth in SEQ ID NO: 47, a LC CDR2 having the amino acid sequence WAF set forth in SEQ ID NO: 48, and a LC CDR3 having the amino acid sequence QQHYRSPWT set forth in SEQ ID NO: 49, or variants of such antibodies comprising 1, 2, 3, 4, 5, or 6 amino acid substitutions in the CDR regions. In one non-limiting embodiment, an anti-TMPRSS6 antibody disclosed herein is the antibody identified herein as the 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.

[0021] In one embodiment, an anti-TMPRSS6 antibody disclosed herein comprises a 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 a 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 a HC CDR1 having the amino acid sequence GFNIEDYY set forth in SEQ ID NO: 52, a HC CDR2 having the amino acid sequence IDPEDAET set forth in SEQ ID NO: 53, a HC CDR3 having the amino acid sequence ARSIYLDPMDY set forth in SEQ ID NO: 54; a LC CDR1 having the amino acid sequence QDVTTA set forth in SEQ ID NO: 57, a LC CDR2 having the amino acid sequence WAT set forth in SEQ ID NO: 58, and a LC CDR3 having the amino acid sequence QQHYSTPYT set forth in SEQ ID NO: 59, or variants of such antibodies comprising 1, 2, 3, 4, 5, or 6 amino acid substitutions in the CDR regions. In one non-limiting embodiment, an anti-TMPRSS6 antibody disclosed herein is the antibody identified herein as the humanized anti-TMPRSS6 antibody variant hzMWTx-003Var, which comprises an HC polypeptide having the amino acid sequence shown in SEQ ID NO:81 and an LC polypeptide having the amino acid sequence shown in SEQ ID NO:83.

[0022] In another aspect, anti-TMPRSS6 antibodies (including variants and fragments as disclosed herein) are provided that can be used to treat iron metabolism disorders, such as iron overload disorders, particularly β-thalassemia and other disorders of ineffective erythropoiesis. Methods and compositions for using the anti-TMPRSS6 antibodies as disclosed herein for therapeutic uses are provided, including but not limited to the treatment of iron metabolism disorders, such as iron overload disorders, particularly β-thalassemia and other disorders of ineffective erythropoiesis. In certain embodiments, pharmaceutical compositions comprising the anti-TMPRSS6 antibodies disclosed herein and suitable carriers and / or excipients are provided.

[0023] In another aspect, methods for treating iron metabolism disorders are provided, such methods comprising administering an effective amount of an anti-TMPRSS6 antibody disclosed herein to a subject in need thereof, wherein administration of the effective amount of the anti-TMPRSS6 antibody modulates the activity of components involved in iron metabolism. In certain embodiments, methods for treating iron overload disorders comprise administering an effective amount of an anti-TMPRSS6 antibody disclosed herein, wherein administration of the effective amount of the anti-TMPRSS6 antibody modulates the activity of components involved in iron metabolism. In certain embodiments, methods for treating iron overload disorders comprise administering an effective amount of an anti-TMPRSS6 antibody disclosed herein, wherein administration of the effective amount of the anti-TMPRSS6 antibody modulates the activity of at least one component involved in regulating hepcidin expression. In certain embodiments, the method comprises administering an effective amount of an anti-TMPRSS6 antibody that inhibits TMPRSS6 suppression of hepcidin expression. In certain embodiments, administration of an effective amount of an anti-TMPRSS6 antibody increases hepcidin expression. In certain embodiments, the method comprises administering an effective amount of an anti-TMPRSS6 antibody that increases the activity of the hepcidin promoter. In certain embodiments, the method comprises administering an effective amount of an anti-TMPRSS6 antibody that inhibits TMPRSS6 repression of BMP / SMAD pathway-induced hepcidin expression. In certain embodiments, the method comprises administering to a subject an effective amount of an anti-TMPRSS6 antibody that results in one or more biological effects associated with an iron overload disorder, including but not limited to reduced serum iron, reduced liver 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 / or increased production of mature red blood cells (increased erythropoiesis).

[0024] In another aspect, methods are provided for treating a disease or condition in which abnormal repression of hepcidin expression is involved, comprising administering an effective amount of an anti-TMPRSS6 antibody disclosed herein to a subject in need thereof, wherein administration of the effective amount of the anti-TMPRSS6 antibody modulates the activity of at least one component involved in abnormal repression of hepcidin expression and reduces abnormal repression of hepcidin expression. In certain embodiments, the method results in increased hepcidin expression.

[0025] In another aspect, methods for treating iron metabolism disorders associated with suppressed hepcidin levels are provided, comprising administering an effective amount of an anti-TMPRSS6 antibody disclosed herein to a subject in need thereof, wherein administration of the effective amount of the anti-TMPRSS6 antibody modulates the activity of at least one component involved in suppressing hepcidin levels. In certain embodiments, the method comprises administering an effective amount of an anti-TMPRSS6 antibody that increases serum hepcidin levels, increases liver hepcidin RNA, and decreases serum iron levels.

[0026] In another aspect, methods for treating iron metabolism disorders are provided, including disorders associated with ineffective erythropoiesis and / or characterized by ineffective erythropoiesis, which may include, but are not limited to, beta-thalassemia. According to this aspect, such methods include administering an effective amount of an anti-TMPRSS6 antibody disclosed herein to a subject known or suspected of having an iron metabolism disorder associated with ineffective erythropoiesis and / or characterized by ineffective erythropoiesis, wherein administration results in one or more changes associated with 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 improves at least one biological effect or symptom associated with the disorder. In specific embodiments, practicing this 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 the production of mature red blood cells (increasing erythropoiesis).

[0027] In another aspect, methods for treating myeloproliferative disorders are provided, 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 clumps and erythroid hyperplasia, polycythemia vera (PV), and / or disorders characterized by polycythemia and erythroid hyperplasia. According to this aspect, such methods comprise administering an effective amount of an anti-TMPRSS6 antibody disclosed herein to a subject known or suspected of having a myeloproliferative disorder. 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 specific embodiments, when administered to a subject known or suspected of having a myeloproliferative disorder, practicing the method results in one or more changes including but not limited to decreased RBC, decreased HCT, decreased hemoglobin (HGB), decreased mean corpuscular volume (MCV), and decreased RDW. In a specific embodiment, when administered to a subject known or suspected of having polycythemia vera (PV), practicing the method results in one or more changes including, but not limited to, decreased RBC, decreased HCT, decreased hemoglobin (HGB), decreased mean corpuscular volume (MCV), and decreased RDW.

[0028] In another aspect, methods for diagnosing or screening for an iron overload disorder in a subject are provided. In certain embodiments, the method comprises administering an anti-TMPRSS6 antibody to a subject known or suspected of having an iron overload disorder and measuring one or more biological effects or symptoms associated with the iron overload disorder.

[0029] In another aspect, methods for diagnosing or screening for a myeloproliferative disorder in a subject are provided. In certain embodiments, the method comprises administering an anti-TMPRSS6 antibody to a subject known or suspected of having a myeloproliferative disorder and measuring one or more biological effects or symptoms associated with the myeloproliferative disorder.

[0030] In another aspect, one or more isolated nucleic acid molecules are provided that encode at least a portion of at least one anti-TMPRSS6 antibody disclosed herein. In certain embodiments, the isolated nucleic acid molecule encoding at least a portion of at least one anti-TMPRSS6 antibody disclosed herein comprises a nucleotide sequence as shown in Table 1, Table 2, or Table 3, or a sequence having substantial identity (e.g., at least 85%, 90%, 92%, 95%, 97%, or 98%, 99% identity) to a nucleotide sequence as shown in Table 1, Table 2, or Table 3. In certain embodiments, the isolated nucleic acid molecule encoding at least one heavy chain (HC) sequence of an anti-TMPRSS6 antibody 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: 79. NO:78, or SEQ ID NO:82 or a sequence substantially identical to SEQ ID NO:82.In certain embodiments, the isolated nucleic acid molecule encoding at least one light chain (LC) sequence 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; or 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: 77 or a sequence substantially identical to SEQ ID NO: 77; or SEQ ID NO: 78 or a sequence substantially identical to SEQ ID NO: 78. NO:80 or a sequence substantially identical to SEQ ID NO:80, or SEQ ID NO:84 or a sequence substantially identical to SEQ ID NO:84.

[0031] In another aspect, a vector comprising one or more nucleic acid molecules encoding at least one amino acid sequence of an anti-TMPRSS6 antibody disclosed herein is provided. In certain embodiments, a vector comprising one or more nucleic acid molecules encoding at least one heavy chain (HC) or light chain (LC) sequence of an anti-TMPRSS6 antibody disclosed herein is provided. In certain embodiments, a vector comprising a nucleic acid molecule encoding at least a portion of at least one amino acid sequence as shown in Table 1, Table 2, or Table 3, or at least a portion of an amino acid sequence having substantial identity to an amino acid sequence as shown in Table 1, Table 2, or Table 3 is provided. In certain embodiments, a vector comprising a nucleic acid molecule encoding at least a portion of at least one HC or LC sequence as shown in Table 1, Table 2, or Table 3, or at least a portion of an amino acid sequence having substantial identity to at least one HC or LC sequence as shown in Table 1, Table 2, or Table 3 is provided.

[0032] In another aspect, at least one host cell is provided that contains a vector comprising one or more nucleic acid molecules encoding an 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 HC or LC sequence as shown in Table 1, Table 2, or Table 3, or at least a portion of an amino acid sequence having substantial identity to at least one HC or LC sequence as shown in Table 1, Table 2, or Table 3. In certain embodiments, the at least one host cell is capable of supporting the expression of the vector and the recombinant production of the anti-TMPRSS6 antibody or antigen-binding fragment thereof encoded by the vector. In certain embodiments, the at least one host cell is capable of supporting the expression of the vector and the recombinant production of the anti-TMPRSS6 antibody or antigen-binding fragment thereof encoded by the vector comprising a nucleic acid molecule encoding at least a portion of at least one HC or LC sequence as shown in Table 1, Table 2, or Table 3, or at least a portion of an amino acid sequence having substantial identity to at least one HC or LC sequence as shown in Table 1, Table 2, or Table 3. In certain embodiments, a host cell is 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, wherein the host cell is capable of supporting expression of the vector and recombinant production of the anti-TMPRSS6 antibody or antigen-binding fragment thereof encoded by the vector.

[0033] In some aspects, the present disclosure provides methods 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 complementary determining region 1 (HC CDR1) comprising the amino acid sequence of SEQ ID NO: 52, an HC CDR2 comprising the amino acid sequence of SEQ ID NO: 53, an HC CDR3 comprising the amino acid sequence of SEQ ID NO: 54, a light chain complementary determining region 1 (LC CDR1) comprising the amino acid sequence of SEQ ID NO: 57, an LC CDR2 comprising the amino acid sequence of SEQ ID NO: 58, and an 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 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. In some embodiments, the antibody comprises 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.In some embodiments, the antibody comprises 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. In some embodiments, the antibody comprises 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.

[0034] In some aspects, the present disclosure provides a method for treating polycythemia vera (PV) in a subject. In some embodiments, the subject has bone marrow containing cells with over-activated JAK2 / STAT5. 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 complementary 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 complementary 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 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. 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, 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.In some embodiments, the antibody comprises 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. In some embodiments, the antibody comprises 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.

[0035] In some embodiments, the subject has a mutation that results in overactivation of JAK2 / STAT5.

[0036] In some embodiments, the subject has a JAK2 mutation.

[0037] 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 homozygous for the JAK2 V617F mutation.

[0038] 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-F547dup, F537-I546dup10F547L, F537IK539I, H538-K539delinsL, H538-K539del, H538DK539LI540S, H538G, K539L, K539E, I540-E543delinsMK, I540-E542delinsS, R541-E543delinsK, N542-E543del, D544-L545del, or S547insLI540-F547dup8.

[0039] In some embodiments, the subject has a JAK2 gene exon 15 mutation. In some embodiments, the JAK2 gene exon 15 mutation is L642P or I645V.

[0040] In some embodiments, the subject has a non-Jak2 mutation. In some embodiments, the subject has a mutation in: 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, 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.

[0041] In some embodiments, the mutation is an acquired mutation, a familial mutation, or a congenital mutation.

[0042] In some embodiments, the subject comprises hematopoietic progenitor cells comprising one or more mutations. In some embodiments, the one or more mutations are in 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 prior to administration.

[0043] In some embodiments, the subject has an increased hematocrit (HCT) relative to a subject without PV. In some embodiments, the subject has splenomegaly prior to administration. In some embodiments, the subject has polycythemia vera prior to administration. In some embodiments, the subject has leukocytosis prior to administration. In some embodiments, the subject has thrombocythemia prior to administration. In some embodiments, the subject has increased hemoglobin relative to a subject without PV prior to administration. In some embodiments, the subject has increased red blood cell distribution width (RDW) relative to a subject without PV prior to administration.

[0044] In some embodiments, the administration of the antibody increases serum hepcidin. In some embodiments, the administration of the antibody reduces liver iron. In some embodiments, the administration of the antibody reduces HCT. In some embodiments, the administration of the antibody reduces red blood cell count. In some embodiments, the administration of the antibody reduces red blood cell distribution width (RDW). In some embodiments, the administration of the antibody reduces serum iron. In some embodiments, the administration of the antibody alleviates leukocytosis. In some embodiments, the administration of the antibody reduces early erythroid progenitor cells. In some embodiments, the administration of the antibody reduces plasma hemoglobin levels. In some embodiments, the administration of the antibody reduces mean corpuscular volume (MCV). In some embodiments, the administration of the antibody reduces the frequency of thrombotic events (TE). In some embodiments, the administration of the antibody reduces the frequency of phlebotomy. In some embodiments, the administration of the antibody reduces the frequency of cytoreductive therapy. In some embodiments, the antibody treatment is provided to a subject in need thereof who is resistant to phlebotomy and / or cytoreductive therapy. In some embodiments, administration of the antibody results in a reduction in symptoms as described by the Myeloproliferative Neoplasm Symptom Assessment Form (MPN-SAF).

[0045] In some embodiments, an antibody is administered to a subject, wherein the antibody comprises a heavy chain complementary 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 complementary 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.

[0046] In some embodiments, the subject is administered an antibody comprising a heavy chain variable region (VH) comprising an amino acid sequence that is 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 that is 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, the subject is administered an antibody comprising a VH comprising an amino acid sequence that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 that is 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, the subject is administered an antibody comprising a VH comprising an amino acid sequence that is 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 that is 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 variation occurs in the framework regions of the VH and / or the framework regions of the VL of the anti-TMPRSS6 antibodies described herein.

[0047] 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.

[0048] In some embodiments, the subject is administered an antibody comprising a heavy chain comprising an amino acid sequence that is 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 that is 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, the subject is administered an antibody comprising a heavy chain comprising an amino acid sequence that is 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 that is 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, the subject is administered an antibody comprising a heavy chain comprising an amino acid sequence that is 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 that is 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, the subject is administered an antibody comprising a heavy chain comprising an amino acid sequence that is 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 that is 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, the subject is administered an antibody comprising a heavy chain comprising an amino acid sequence that is 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 that is 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, the subject is administered an antibody comprising a heavy chain comprising an amino acid sequence that is 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 that is 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 variation occurs in the framework region of the VH, framework region of the VL, heavy chain constant region, and / or light chain constant region of the anti-TMPRSS6 antibodies described herein.

[0049] In some embodiments, 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 is administered to the subject.

[0050] 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.

[0051] In some embodiments, the subject receives one or more additional therapeutic agents for the treatment of PV. In some embodiments, the additional therapeutic agents for the treatment of PV include interferons (e.g., Ropeinterferon alpha-2b-njft (Besremi), pegylated interferon), JAK2 inhibitors (e.g., Ruxolitinib, XL019, Fidatinib (SAR302503), Molotinib), JAK1 inhibitors (e.g., Itatinib), hepcidin mimetics (e.g., Rusfitide (PTG-300)), lysine-specific demethylase inhibitors (e.g., Bomedemstat (IMG-7298), TMPRSS6 antagonists (e.g., Sapablursen (ISIS 702843), SLN124), anti-TfR1 antibodies (e.g., PPMX-T003), MDM2 inhibitors (e.g., edanuline (RG7388), KRT-232), tyrosine kinase inhibitors (e.g., dasatinib, erlotinib, Gleevec, lestaurinib (CEP-701)), HDAC inhibitors (e.g., gilvestrastat (ITF2357), MK-0683), PI3K inhibitors (e.g., erbulisam (TGR-1202), telomerase inhibitors (e.g., imetelstat), phlebotomy, low-dose aspirin, or hydroxyurea. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 shows the results from a cascade screen of anti-TMPRSS6 antibodies, in which antibodies that bound to human TMPRSS6 were evaluated for HAMP promoter activity using an in vitro functional assay, and antibodies that showed an effect on HAMP promoter activity were evaluated for cross-reactivity with non-human TMPRSS6.

[0054] Figures 2A-2F Shown are the effects of anti-TMPRSS6 antibodies on HAMP promoter activity measured by dual-luciferase reporter assay in HepG2 cells for a range of antibody concentrations. In each graph, open circles represent the results using anti-TMPRSS6 antibodies, while open squares represent the results using the same concentrations of mouse IgG or human IgG1 as negative (non-specific binding) controls. Figure 2AShown are the effects of MWTx-001 anti-TMPRSS6 antibody on HAMP promoter activity over a range of antibody concentrations. Figure 2B Shown are the effects of the MWTx-002 anti-TMPRSS6 antibody on HAMP promoter activity over a range of antibody concentrations. Figure 2C Shown are the effects of MWTx-003 anti-TMPRSS6 antibody on HAMP promoter activity over a range of antibody concentrations. Figure 2D Shown are the effects of hzMWTx-001Var anti-TMPRSS6 antibody on HAMP promoter activity over a range of antibody concentrations. Figure 2E Shown are the effects of hzMWTx-002Var anti-TMPRSS6 antibody on HAMP promoter activity over a range of antibody concentrations. Figure 2F Shown are the effects of hzMWTx-003Var anti-TMPRSS6 antibody on HAMP promoter activity over a range of antibody concentrations.

[0055] Figures 3A-3M Shown are the results of determining the binding affinity of anti-TMPRSS6 antibodies. Figures 3A-3F Figure 2 shows the results of determining the binding affinity of anti-TMPRSS6 antibodies for human TMPRSS6 expressed on HEK293T cells using two different methods. In each figure, open circles represent the results using anti-TMPRSS6 antibodies at a range of concentrations, while open squares represent the results using mouse IgG at the same concentrations as a negative control. Figures 3A-3C The results show that MWTx-001 ( Figure 3A )、MWTx-002( Figure 3B ) and MWTx-003( Figure 3C ) and human TMPRSS6, where the EC values ​​calculated for each antibody were 50 The values ​​were used as estimates of binding affinity. Figures 3D-3F The results show that the expression of MWTx-001 ( Figure 3D )、MWTx-002( Figure 3E ) and MWTx-003( Figure 3F ) and human TMPRSS6, where the EC values ​​calculated for each antibody were 50 The values ​​were used as estimates of binding affinity. Figure 3G-3M Shows the use RED96e, determination of affinity and binding kinetics of anti-TMPRSS6 antibodies for human etco-TMPRSS6-FLAG using 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 Shown are the binding kinetics of the MWTx-001 anti-TMPRSS6 antibody to etco-TMPRSS6-FLAG. Figure 3H Shown are the binding kinetics of the MWTx-002 anti-TMPRSS6 antibody to etco-TMPRSS6-FLAG. Figure 3I Shown are the binding kinetics of the MWTx-003 anti-TMPRSS6 antibody to etco-TMPRSS6-FLAG. Figure 3J Shown are the binding kinetics of hzMWTx-001Var anti-TMPRSS6 antibody to etco-TMPRSS6-FLAG. Figure 3K Shown are the binding kinetics of hzMWTx-002Var anti-TMPRSS6 antibody to etco-TMPRSS6-FLAG. Figure 3L Shown are the binding kinetics of hzMWTx-003Var anti-TMPRSS6 antibody to etco-TMPRSS6-FLAG. Figure 3M Affinity measurements of all anti-TMPRSS6 antibodies are summarized.

[0056] Figures 4A-4U Shown are the results of determining the cross-reactivity of anti-TMPRSS6 antibodies. Figures 4A-4I The results of the determination of the cross-reactivity of anti-TMPRSS6 antibodies MWTx-001, MWTx-002 and MWTx-003 with human TMPRSS6 and non-human TMPRSS6 expressed on HEK293T cells are shown. Each histogram shows the FACS results for a single antibody incubated with HEK293T cells expressing the TMPRSS6 target (thin line and light fill; indicated by the antibody name) and the same antibody incubated with control HEK293T cells that do not express the TMPRSS6 protein (thick line, dark fill; indicated by Ctrl). Figures 4A-4C The results show that HEK293T cells stably expressing human TMPRSS6 (HuTMPRSS6-(His)6) and MWTx-001 ( Figure 4A )、MWTx-002( Figure 4B ) and MWTx-003( Figure 4C ) results. Figures 4D-4F HEK293T cells stably expressing mouse TMPRSS6 (MoTMPRSS6-(His)6) and MWTx-001 ( Figure 4D )、MWTx-002( Figure 4E ) and MWTx-003( Figure 4F ) results. Figures 4G-4I HEK293T cells transiently expressing cynomolgus monkey TMPRSS6 (CynoTMPRSS6-(His)6) and MWTx-001 ( Figure 4G )、MWTx-002( Figure 4H ) and MWTx-003( Figure 4I ) results. Figures 4J-4U The results show that the MWTx-001 anti-TMPRSS6 antibody ( Figures 4J-4K )、MWTx-002 anti-TMPRSS6 antibody ( Figures 4L-4M )、MWTx-003 anti-TMPRSS6 antibody ( Figure 4N-4O )、hzMWTx-001Var anti-TMPRSS6 antibody ( Figure 4P-4Q )、hzMWTx-002Var anti-TMPRSS6 antibody ( Figure 4R-4S ) and hzMWTx-003Var anti-TMPRSS6 antibody ( Figure 4T-4U ) and non-human TMPRSS6, anti-TMPRSS6 antibodies bind to non-human (mouse ( Figure 4J 、 4L , 4N, 4P, 4R, 4T) or cynomolgus monkeys ( Figure 4K 、 4M , 4O, 4Q, 4S, 4U)) results of cross-reactivity of TMPRSS6. In each figure, open circles represent the results using anti-TMPRSS6 antibodies, and open squares represent the results of mouse IgG or human IgG1 as negative (non-specific binding) controls, where the EC values ​​calculated for each antibody are 50 The values ​​were used as estimates of binding affinity.

[0057] Figures 5A-5R The anti-TMPRSS6 monoclonal antibody MWTx-001 ( Figures 5A-5C )、MWTx-002( Figures 5D-5F )、MWTx-003( Figures 5G-5I ) anti-TMPRSS6 antibody and its humanized variant hzMWTx-001Var( Figures 5J-5L )、hzMWTx-002Var( Figures 5M-5O )、hzMWTx-003Var( Figure 5P-5R) FACS analysis of the binding of anti-TMPRSS6 antibodies to HEK293T cells expressing homologous matriptases. HEK293T cells stably expressing human TMPRSS6 (matriptase-2) ( Figure 5A 、 5D , 5G, 5J, 5M, 5P) were used as positive controls, while overexpression of matriptase (ST14) ( Figure 5B 、 5E , 5H, 5K, 5N, 5Q) and / or matriptase-3 (TMPRSS7) ( Figure 5C 、 5F HEK293T cells expressing 5I, 5L, 5O, and 5R proteins were used to test their binding to the homologous matriptase. Figures 5A-5R ), HEK293T cells not expressing matriptase (HEK293T) were used as a negative control, where the control (Ctrl) results are clearly indicated.

[0058] Figures 6A-6L It was shown that anti-TMPRSS6 antibody treatment increased hepcidin expression in mice in a dose-dependent manner. Figures 6A-6C MWTx-003 anti-TMPRSS6 antibody ( Figures 6A-6B ) or its humanized variant hzMWTx-003Var anti-TMPRSS6 antibody ( Figure 6C ) on serum iron. Figure 6D The effect of GFP-TMPRSS6 on serum hepcidin is shown. Figures 6D-6F MWTx-003 anti-TMPRSS6 antibody ( Figures 6D-6E ) or its humanized variant hzMWTx-003Var anti-TMPRSS6 antibody ( Figure 6F ) on serum hepcidin. Figure 6G Shown is the effect of GFP-TMPRSS6 on hepatic hepcidin RNA. Figures 6G-6I 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 MWTx-003 anti-TMPRSS6 antibody ( Figures 6J-6K ) or its humanized variant hzMWTx-003Var anti-TMPRSS6 antibody ( Figure 6L ) serum concentration. Mouse IgG2b (MoIG2b) ( Figures 6A-6B , 6D-6E, 6G-6H, 6J-6K) or human IgG1 (HuIGg1) ( Figure 6C 、 6F , 6I, 6L) were used as isotype controls, PBS was used as vehicle control, and GFP vector was used as vector control ( Figure 6A 、 6D , 6G, 6J).

[0059] Figures 7A-7R In vivo efficacy of anti-TMPRSS6 antibodies using a β-thalassemia mouse model was shown. Figures 7A-7D Using Th3 / + mice, MWTx-003 anti-TMPRSS6 antibody showed the effect of anti-TMPRSS6 antibody on RBC ( Figure 7A )、HGB( Figure 7B )、HCT( Figure 7C ) and RDW( Figure 7D )’s role. Figure 7E Shown are the effects of MWTx-003 anti-TMPRSS6 antibody on spleen weight using Th3 / + mice. Figure 7F Shown are the effects of MWTx-003 anti-TMPRSS6 antibody on serum iron using Th3 / + mice. Figure 7G Shown are the effects of the MWTx-003 anti-TMPRSS6 antibody on liver non-heme iron using Th3 / + mice. Figure 7H Shown are the effects of MWTx-003 anti-TMPRSS6 antibody on serum hepcidin using Th3 / + mice. Figure 7I Shown is the effect of MWTx-003 anti-TMPRSS6 antibody on hepatic hepcidin RNA using Th3 / + mice. Figure 7J Shown are serum concentrations of MWTx-003 anti-TMPRSS6 antibody using Th3 / + mice. Figures 7L-7M Shown are the effects of the MWTx-003 anti-TMPRSS6 antibody on erythropoiesis using bone marrow from Th3 / + mice. Figures 7O-7P Shown are the effects of the MWTx-003 anti-TMPRSS6 antibody on erythropoiesis using splenocytes from Th3 / + mice. Figures 7K-7P Representative figures in Figure 4 show four different cell clusters (I: basophilic erythroblasts; II: polychromatic erythroblasts; III: normochromatic erythroblasts and anucleated reticulocytes, and IV: mature erythrocytes) and highlight their corresponding percentages of cell numbers. Wild-type mice were used as positive controls ( Figures 7A-7J , 7K, 7N), while mouse IgG2b (MoIgG2b) was used as an isotype control in treatment ( Figures 7A-7J , 7L, 7O). Figures 7Q-7RThe bar graph in Figure 4 shows the bone marrow ( Figure 7Q ) and spleen ( Figure 7R ) averaged results for cell clusters I, II, III, and IV in the 3D-staining chromatin matrix, comparison of which allowed identification of shifts in each population following MWTx-003 treatment, most notably toward mature red blood cells (cluster IV).

[0060] Figures 8A-8D Shows the use Epitope binning results of RED96e, MWTx-001, MWTx-002 and MWTx-003 anti-TMPRSS6 antibodies on human etco-TMPRSS6-FLAG. Figure 8A Epitope grouping of the MWTx-001 anti-TMPRSS6 antibody against etco-TMPRSS6-FLAG is shown. Figure 8B Epitope grouping of the MWTx-002 anti-TMPRSS6 antibody against etco-TMPRSS6-FLAG is shown. Figure 8C Epitope grouping of the MWTx-003 anti-TMPRSS6 antibody against etco-TMPRSS6-FLAG is shown. Figure 8D The binding signals for MWTx-001, MWTx-002, and MWTx-003 anti-TMPRSS6 antibodies are summarized.

[0061] Figures 9A-9H Shown are the results of subchronic treatment with anti-TMPRSS6 antibodies in the Jak2V617 / +Vav-iCre mouse model of PV when 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 were not treated; each symbol in the figure represents a single mouse. Figures 9A-9C Hematological parameters HCT ( Figure 9A )、RBC( Figure 9B ) and HGB( Figure 9C ) endpoint measurement. Figures 9D-9E Endpoint measures for each treatment and dose level are also shown, where Figure 9D Splenomegaly (Splenomegaly Index measured as mg / g body weight) is shown, indicating a dose-dependent development of iron-restricted erythropoiesis in mice treated with MWTx-003. Figure 9ESerum hepcidin levels (ng / ml) are shown, while Figure 9F Shown are serum anti-TMPRSS6 concentrations (μg / ml) measured by cell surface ELISA at the end of the study. Figure 9G Shown are FACS results measuring early erythroid precursors (cluster I, basophilic erythroblasts and cluster II, polychromatic erythroblasts) in the bone marrow (upper row) and spleen (lower row), showing results for WT (left panel, upper and lower), MoIgG2b isotype control (middle panel, upper and lower), and treatment with 10 mg / kg of anti-TMPRSS6 MWTx-003 (right panel, upper and lower). Figure 9H Prussian blue stained images of liver sections (left panel) and spleen sections (right panel) from mouse IgG2b isotype control MoIgG2b treatment (top row) and increasing doses of anti-TMPRSS6 MWTx-003 are shown, indicating increased iron deposition in the spleen, but no significant changes in liver iron content between treated mice relative to controls. Significant iron deposition is indicated by arrows. Figures 9A-9F One-way ANOVA with Tukey's multiple comparison adjustment was used, ****P<0.0001, ***P<0.001, *P<0.05. Detailed Description of the Invention

[0063] The present invention relates to novel antibodies and antigen-binding fragments thereof that bind to TMPRSS6, as well as methods of making and using the same.

[0064] I. Terms / Definitions

[0065] Unless otherwise defined, the scientific and technical terms used in conjunction with the present invention should have the meanings commonly understood by those of ordinary skill in the art. The use of singular terms ("a" or "an" or "the" or other uses of singular terms) includes plural referents, and plural terms should include the singular unless the context clearly indicates otherwise. Thus, for example, reference to "an antibody" includes "one or more" antibodies or "a plurality" of such antibodies. All publications mentioned herein are hereby incorporated by reference in their entirety.

[0066] Generally, the nomenclature and technology of molecular biology, microbiology, cell and tissue culture, protein and nucleotide chemistry available to those skilled in the art and recombinant DNA technology can be used for antibody disclosed herein, Fab, composition and method.Technology and procedure described herein are generally according to conventional methods well known in the art, and as various general and more specific execution described in reference, described reference is especially Sambrook et al. (1989) MOLECULAR CLONING:A LABORATORY MANUAL (second edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) and Ausubel et al. (1994) CURRENT PROTOCOLS INMOLECULAR BIOLOGY, I-III volumes (John Wiley & Sons, NY). Unless otherwise indicated herein, enzymatic reaction and purification technique are according to the instructions of manufacturers or as generally completed in this area or as described herein. Conventional nomenclature is used herein to describe the technology and method for drug preparation and preparation and the treatment of experimenter.

[0067] "Antibody" in the broadest sense refers to a polypeptide or combination of polypeptides that recognizes and binds an antigen through one or more immunoglobulin variable regions, where the immunoglobulin variable region(s) may be naturally occurring or non-naturally occurring, e.g., as a result of engineering, chimerization, humanization, optimization, CDR grafting, or affinity maturation.

[0068] As disclosed herein, an "antibody" 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 comprises at least sufficient complementarity determining regions (CDRs) interspersed with framework regions (FRs) for the antibody to recognize and bind to an antigen. The anti-TMPRSS6 antibodies disclosed herein may be, but are not limited to, at least one of the following: 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 attached to the antibody and / or additional amino acids added to the N-terminus or C-terminus, or other TMPRSS6 binding fragments or variants. Whole antibodies, full-length antibodies, intact antibodies, naturally occurring antibodies, or equivalent terms are understood to refer to polypeptides, particularly glycoproteins, 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 a LC constant region (CL). The HC and LC variable regions, VH and VL, include binding domains that interact with antigens. The VH and VL regions can be further subdivided into CDR regions characterized by high variability, interspersed with FR regions that are generally more conserved. Each VH and VL is generally composed of three CDRs and four FRs arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant region 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 heavy chain (HC) CDR1, CDR2 and CDR3 and light chain (LC) CDR1, CDR2 and CDR3 sequences, any of which may be naturally occurring or non-naturally occurring. An antibody may contain fewer CDR sequences as long as the antibody can recognize and bind to the antigen.

[0069] The anti-TMPRSS6 antibodies disclosed herein may be variants comprising at least one altered CDR or framework sequence, wherein the CDR and / or framework sequence may be optimized by mutating the nucleic acid molecules encoding such framework sequences. Variants may be constructed with HC and LC portions independently derived from different sources. Techniques for generating variants include, but are not limited to, conservative amino acid substitutions, computer modeling, screening of candidate polypeptides alone or in combination, and codon optimization, and it is understood that a skilled artisan will be able to generate antibody variants as needed. The anti-TMPRSS6 antibodies disclosed herein may be fragments. The antigen-binding function of an antibody may be performed by fragments such as: Fab fragments; monovalent fragments consisting of VL, VH, CL, and CH1 domains; F(ab)2 fragments; bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; Fd fragments consisting of VH and CH1 domains; single-chain variable fragments (scFv) consisting of the VL and VH domains of a single arm of an antibody; single-domain antibody (dAb) fragments consisting of a VH domain; and isolated CDRs (VHH, nanobodies) or aptamers. The antigen binding portion can be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs and bis-scFv (bis-scFv) (see, e.g., 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 monomer (monobodies) (see, e.g., U.S. Patent No. 6,703,199, which describes a fibronectin polypeptide monomer).

[0070] The term antibody encompasses polypeptides of various broad classes that can be distinguished biochemically. " Classification " of antibody refers to the type of constant domain or constant region possessed by its heavy chain. It will be appreciated by those skilled in the art that there are five main classes of antibody, i.e. IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, which are fully characterized and known to confer functional specialization. These classifications and modified forms of isotypes are easily distinguishable and within the scope of the present disclosure. Although all immunoglobulin classes are within the scope of the present disclosure, the present disclosure relates to the IgG classification of immunoglobulin molecules to a great extent.

[0071] The term "chimeric" antibody refers to one in which a portion of the heavy chain (HC) and / or light chain (LC) involved in forming the immunoreactive site is derived from a particular source or species, while the remainder of the HC and / or LC is derived from a different source or species. In certain embodiments, the target binding region or site will be from a non-human source (e.g., mouse or non-human primate), and the constant region is human.

[0072] As used herein, the phrase "humanized antibody" refers to an antibody or antibody variant derived from a non-human antibody, typically a mouse monoclonal antibody, in which the CDRs from the parent non-human antibody are transplanted (fused) into a framework comprising a variable region derived from a human immunoglobulin framework, particularly an acceptor human framework or a human consensus framework. The techniques and principles for designing, preparing 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 29-6 4; 321(6069):522-5; Almagro JC, Fransson J.Humanization of antibodies. Front Biosci. 2008 January 1; 13:1619-33). It will be appreciated that changes can be made to the receptor framework at multiple locations in order to develop humanized antibodies with improved characteristics depending on the desired use, such as high affinity for the target, low clearance, low toxicity, etc. The anti-TMPRSS6 antibodies disclosed herein may be humanized variants.

[0073] "Affinity" refers to the total strength 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, binding affinity as used herein refers to 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 which approximately 50% of maximal binding corresponds (the calculated EC 50 ) can be used as an estimate of affinity. The affinity of a molecule X for its partner Y can generally be measured by the dissociation constant (Kd or KD, representing the k value measured for the interaction). off / k on )express.

[0074] A "subject" is a mammal, wherein mammals include, but are not limited to, primates (e.g., humans and non-human primates, such as monkeys), domestic animals (e.g., cattle, sheep, cats, dogs, pigs, llamas, and horses), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the subject is a human. The phrase "a subject in need thereof" or "a patient in need thereof" or "a patient in need of treatment" or "a subject in need of treatment" may include subjects who benefit from the administration of an anti-TMPRSS6 antibody disclosed herein for the treatment of an iron overload disorder. It should be understood that administration of an anti-TMPRSS6 antibody encompasses administration to a "subject in need thereof," which can be interpreted to refer to a subject who is known or suspected of having an iron overload disorder, particularly β-thalassemia, based on indicators such as symptoms, family history, or genotype. It should be further understood that anti-TMPRSS6 antibodies can be administered to subjects who are not known or suspected of having an iron metabolism disorder for purposes that may include, but are not limited to, preventive or prophylactic purposes, for screening, diagnosis, research purposes, or for purposes of achieving an outcome other than treating a disorder.

[0075] For example, an "effective amount" of an anti-TMPRSS6 antibody in a pharmaceutical formulation refers to an amount effective to achieve the desired therapeutic or preventive result at the necessary dosage and time period. It should be understood that an "effective amount" is intended to refer to an amount of an anti-TMPRSS6 antibody or a pharmaceutical composition comprising an anti-TMPRSS6 antibody that will elicit a biological response in a cell, tissue, system, non-human animal subject, non-human mammalian subject, or human subject to be measured or a desired therapeutic effect thereon. 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 the active agent in the bloodstream or target tissue. The exact amount will depend on a variety of 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, considerations such as the disease state, age, sex, and weight of the subject, and the like, and can be readily determined by one skilled in the art based on the information provided herein or otherwise available in the relevant literature. As used in this context, the terms "improve," "increase," or "decrease" indicate a value or parameter relative to a baseline measurement, e.g., a measurement in the same subject before initiation of a treatment described herein, or a measurement in a control individual (or multiple control individuals) in the absence of a treatment described herein.

[0076] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a formulation that is in such a form as to allow the biological activity of the active ingredient (particularly an anti-TMPRSS6 antibody) contained therein to be effective. It should be understood that a pharmaceutical composition may contain more than one active ingredient, for example, more than one anti-TMPRSS6 antibody, or a combination of an anti-TMPRSS6 antibody and another active ingredient that acts on a different target, wherein such a combination may be, but is 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 that targets a TGF superfamily ligand to stimulate erythropoiesis. A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation other than an active ingredient that is non-toxic to a subject. It should be understood that a pharmaceutically acceptable carrier may be, but is not limited to, a buffer, an excipient, a stabilizer, an adjuvant, or a preservative.

[0077] As used herein, the term "Janus kinase 2 / signal transducer and activator of transcription 5 (JAK2 / STAT5) pathway" or "JAK2 / STAT5 signaling" refers to a cellular signaling pathway (e.g., involved in processes such as immunity, cell division, cell death, and tumor formation) mediated by Janus kinase 2 (JAK2), signal transducer and activator of transcription 5 (STAT5), receptors associated with JAK2, and other regulatory proteins. JAK2 is a member of the Janus kinase family, which are non-receptor protein tyrosine kinases. JAKs (e.g., JAK2) bind to the cytoplasmic domain of several cytokine receptors (e.g., receptors lacking intrinsic kinase activity, 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)). After cytokine binding, JAK2 is activated by tyrosine phosphorylation of the cytoplasmic domain of cytokine receptor.JAK2 activation promotes transcription factor signal transducers and transcription activators (e.g., STAT5) to be recruited to receptor complexes. (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 STAT (e.g., STAT5), and the STAT phosphorylation mediated by JAK2 leads to the formation of stable homodimers or heterodimers (e.g., STAT5 homodimers) of STAT. Phosphorylated STAT dimers (e.g., STAT5 homodimers) translocate to the nucleus and bind to the promoters of downstream genes to start transcription of these genes.

[0078] In some embodiments, JAK has a pseudokinase domain (i.e., JH2 domain) that is located upstream of the C-terminal tyrosine kinase domain (i.e., JH1 domain). In some embodiments, the pseudokinase domain of JAK is critical for maintaining low basal (e.g., in the absence of cytokines) levels of tyrosine kinase activity (see, e.g., Hubbard, Mechanistic Insights into Regulation of JAK2 Tyrosine Kinase, Frontiers in Endocrinology, January 2018, Vol. 8, Article 361).

[0079] As used herein, the term "overactivation of the JAK2 / STAT5 pathway" or "JAK2 / STAT5 overactivation" refers to abnormal activation (e.g., increased activation, constitutive activation) of the JAK2 / STAT5 pathway compared to physiologically normal levels of activation. 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 normal subjects. For example, in some embodiments, overactivation of JAK2 / STAT5 can be determined by comparing JAK2 / STAT5 activity in cells of subjects with mutant JAK2 alleles with JAK2 / STAT5 activity in normal subjects with only wild-type JAK2 alleles, for the purpose of determining whether JAK2 mutations are associated with overactivation. In some embodiments, similar analyses may 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, such as a kinase activity assay (e.g., by BPS Bioscience's JAK2 assay kit catalog #79520; by Eurofins' eXpress EpoR-JAK2 Functional Assay Catalog No. 93-0900E3CP; Jak2 Kinase Assay Kit, catalog number 7752, etc.); Western blotting (e.g., Western blotting for phosphorylated JAK2, STAT5, and / or downstream proteins); RT-PCR (e.g., RT-PCR for expression levels of downstream genes).

[0080] In some embodiments, the subject is a subject with polycythemia vera (PV). In some embodiments, PV is associated with overactivation of JAK2 / STAT5. In some embodiments, the JAK2 / STAT5 activity of a subject with PV 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 overactivation of JAK2 / STAT5. In some embodiments, a subject with polyps has JAK2 / STAT5 activity that is at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 50 times, at least 75 times, at least 100 times, or more than the JAK2 / STAT5 activity of a subject without polyps. In some embodiments, a subject with polyps has JAK2 / STAT5 activity that is at most 1.5 times, at most 2 times, at most 3 times, at most 4 times, at most 5 times, at most 6 times, at most 7 times, at most 8 times, at most 9 times, at most 10 times, at most 15 times, at most 20 times, at most 25 times, at most 50 times, at most 75 times, at most 100 times the JAK2 / STAT5 activity of a subject without polyps.In some embodiments, a subject with PV has 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 9 ...90-fold, 60% to 100-fold, % to 90 times, 70% to 90 times, 80% to 90 times, 90% to 90 times, 1 to 90 times, 5 to 90 times, 10 to 90 times, 20 to 90 times, 25 to 90 times, 50 to 90 times, 75 to 90 times, 10% to 80 times, 20% to 80 times, 30% to 80 times, 40% to 80 times, 50% to 80 times, 60% to 80 times, 70% to 80 times, 80% to 80 times, 90% to 80 times, 1 to 80 times, 5 to 80 times, 10 to 80 times, 20 to 80 times, 25 to 80 times, 50 to 80 times, 75 to 80 times, 10% to 50 times, 20% to 50 times, 30% to 50 times, 40% to 50 times, 50% to 50 times, 60% to 50 times, 70% to 50 times, 80% to 50 times, 90% to 50 times, 1 to 50 times, 5 to 50 times, 10 to 50 times, 20 to 50 times, 25 to 50 times, 10% to 25 times, 20% to 25 times, 30% to 25 times, 40% to 25 times, 50% to 25 times, 60% to 25 times, 70% to 25 times, 80% to 25 times, 90% to 25 times, 1 to 25 times, 5 to 25 times, 10 to 25 times, 20 to 25 times, 2 to 25 times, 12 to 25 times, 18 to 25 times, 10% to 10 times, 20% to 10 times, 30% to 10 times, 40% to 10 times, 50% to 10 times, 60% to 10 times, 7 10-fold, 0% to 10-fold, 80% to 10-fold, 90% to 10-fold, 1 to 10-fold, 5 to 10-fold, 2 to 10-fold, 3 to 10-fold, 4 to 10-fold, 5 to 10-fold, 6 to 10-fold, 7 to 10-fold, 8 to 10-fold, 9 to 10-fold, 10% to 5-fold, 20% to 5-fold, 30% to 5-fold, 40% to 5-fold, 50% to 5-fold, 60% to 5-fold, 70% to 5-fold, 80% to 5-fold, 90% to 5-fold, 1 to 5-fold, 2 to 5-fold, 3 to 5-fold, 4 to 5-fold, 10% to 2-fold, 20% to 2-fold, 30% to 2-fold, 40% to 2-fold, 50% to 2-fold, 60% to 2-fold, 70% to 2-fold, 80% to 2-fold, 90% to 2-fold, or 1 to 2-fold.

[0081] As used herein, the term "treat" or "treating" or similar terms may refer to an outcome that is considered beneficial for a particular subject under defined circumstances. Treating an iron metabolism disorder may nonexclusively refer to any of reducing, ameliorating, slowing, interrupting, preventing, alleviating, stopping, or reversing the progression or severity of an existing symptom, disorder, condition, or disease, and may further encompass preventing or delaying the onset of one or more symptoms of an iron overload disorder, and / or reducing the severity or frequency of one or more symptoms of an iron overload disorder. The term "treat" or "treatment method" or equivalents may encompass one or more uses of the anti-TMPRSS6 antibodies disclosed herein, including but not limited to therapeutic, prophylactic, preventive, diagnostic, imaging, and screening uses.

[0082] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating nucleic acids to which the vector sequences are linked in a host cell into which the vector is introduced. Vectors capable of directing the expression of nucleic acids to which they are operatively linked are referred to herein as "expression vectors."

[0083] II. Anti-TMPRSS6 Antibodies

[0084] Antibodies and antigen-binding fragments are provided that are capable of binding to TMPRSS6 on the surface of a cell and modulating the activity of at least one component involved in iron metabolism, particularly at least one component involved in iron overload disorders associated with abnormal repression of hepcidin expression. Anti-TMPRSS6 antibodies that are capable of binding to TMPRSS6 on the surface of a cell and modulating the activity of at least one component involved in regulating hepcidin expression can be used in methods for treating iron overload disorders associated with abnormal repression of hepcidin expression. Anti-TMPRSS6 antibodies that are capable of binding to TMPRSS6 on the surface of a cell and modulating TMPRSS6 repression of hepcidin expression can be used to therapeutically target TMPRSS6 in methods for treating iron overload disorders and / or other iron disorders and / or disorders associated with abnormal repression of hepcidin expression.

[0085] Once antibodies or fragments specific for TMPRSS6, particularly human TMPRSS6 expressed on the surface of a cell, have been obtained, they can be tested for the desired biological activity of modulating the activity of at least one component involved in iron metabolism by several methods known to the skilled artisan.

[0086] It should be understood that, as used herein, "modulate" or "modulating" or similar terms can refer to one or more effects that can occur when the anti-TMPRSS6 antibodies disclosed herein bind to their targets. Depending on the component in question, "modulate" and its equivalents can refer to different modes of action and effects, that is, 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 agonist effect on these components.

[0087] It should be 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. Therefore, a component within the meaning of a process or pathway can be, but is not limited to, regulation of hepcidin expression, TMPRSS6 suppression of hepcidin expression, a process of hepcidin expression, regulation of hepcidin levels, increasing hepcidin levels, activity of the hepcidin promoter, or TMPRSS6 suppression of hepcidin expression induced by the BMP / SMAD pathway, regulation of liver non-heme iron levels, one or more processes involving splenomegaly, or one or more hematopoietic processes involving red blood cell count (RBC), hematocrit (HCT), red blood cell distribution width (RDW), and erythropoiesis regulation, in particular the production of mature red blood cells.

[0088] Anti-TMPRSS6 antibodies as disclosed herein can be used to therapeutically target at least one component involved in iron metabolism, in particular at least one component involved in iron overload disorders. In certain embodiments, anti-TMPRSS6 antibodies as 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 an increase in hepcidin expression. In certain embodiments, anti-TMPRSS6 antibodies as disclosed herein can be used to modulate the activity of the hepcidin promoter to achieve an increase in hepcidin expression. It should be understood that anti-TMPRSS6 antibodies as disclosed herein can be used to therapeutically target TMPRSS6 and thereby modulate the downstream activity of other components of hepcidin expression, including but not limited to regulation of liver non-heme iron levels, one or more processes involved in splenomegaly, or one or more hematopoietic processes involved in red blood cell count (RBC), hematocrit (HCT), red blood cell distribution width (RDW), and erythropoiesis regulation, in particular the production of mature red blood cells.

[0089] Therapeutic targeting of at least one component involved in iron metabolism using anti-TMPRSS6 antibodies as disclosed herein allows for precise regulation of the targeted component. It will be appreciated that by precisely targeting TMPRSS6 and its downstream effects on at least one component involved in regulating hepcidin expression using anti-TMPRSS6 antibodies as disclosed herein, it is possible to avoid the undesirable effects, difficulties with delivery and / or efficacy, and regulatory hurdles associated with other approaches currently in use or under development for treating iron overload disorders, such as blood transfusions that may further exacerbate iron overload, iron chelation with poor patient compliance, invasive phlebotomy or splenectomy for symptomatic treatment only, gene therapy targeting the HBB gene with potentially permanent pleiotropic effects in multiple systems, gene therapy and gene editing with unknown off-target effects, Fc fusion proteins targeting TGF superfamily ligands to inhibit SMAD signaling, which do not reduce the need for iron chelation therapy for iron overload, and other approaches that are difficult to control or deliver, such as hepcidin mimetics and antisense or iRNA drugs targeting TMPRSS6. It should be understood that the use of anti-TMPRSS6 antibodies for precision therapeutic targeting does not preclude the possibility of using anti-TMPRSS6 antibodies in methods and compositions for combination therapy, for example, in combination with another active ingredient that acts on a different target, in combination with antibodies that bind to a different target, in combination with gene therapy agents and methods for targeting the HBB gene, or in combination with Fc fusion proteins that target TGF superfamily ligands to stimulate erythropoiesis.

[0090] The anti-TMPRSS6 antibodies disclosed herein allow for the development of treatments that can be customized for each subject (e.g., dosage, frequency of administration), where they can be easily continued and discontinued, and combined with other therapies. In certain strategic embodiments, the anti-TMPRSS6 antibodies disclosed herein can be combined with other therapies that may target multiple therapeutic targets and / or address deficiencies or undesirable effects of one of the therapies in the combination therapy.

[0091] Exemplary embodiments of anti-TMPRSS6 antibodies and uses thereof

[0092] Non-limiting exemplary embodiments of the anti-TMPRSS6 antibodies of the invention are disclosed herein, particularly in the Examples, Tables, and Figures.

[0093] a. Antibodies that bind to TMPRSS6

[0094] As demonstrated in the Examples, a functional cascade can be used to identify and characterize the anti-TMPRSS6 antibodies of the present invention, wherein the first step in the cascade involves screening for antibodies capable of binding to human TMPRSS6 on the surface of cells expressing TMPRSS6 (Example 1, Figure 1), followed by a second step of identifying antibodies capable of binding to human TMPRSS6 on the surface of cells expressing TMPRSS6 and modulating the activity of components involved in iron metabolism, in this case, testing for the ability to increase hepcidin (HAMP) promoter activity (Example 2). As demonstrated by the exemplary embodiment shown in Figure 1, the first step identified 143 antibodies (clones) capable of binding to human TMPRSS6 on the surface of cells expressing TMPRSS6, and the second step identified ten (10) antibodies (from the 143 antibodies screened) as "active" antibodies (clones) capable of increasing hepcidin (HAMP) promoter activity.

[0095] In the third step of the functional cascade ( FIG1 ), ten (10) "active" antibodies were tested for cross-reactivity with non-human TMPRSS6 targets from sources relevant for further studies, i.e., cross-reactivity with mouse TMPRSS6 in a mouse model relevant for preclinical efficacy studies, and cross-reactivity with cynomolgus monkey TMPRSS6 in relevant for toxicity (safety) trials. As demonstrated by the exemplary embodiment shown in FIG1 , three (3) clones (out of the 10 screened) showed cross-reactivity with at least one non-human TMPRSS6, as demonstrated in Example 4 and shown in FIG4 , and were designated MWTx-001, MWTx-002, and MWTx-003. Each monoclonal antibody was sequenced, and the CDRs (Kabat numbering) on ​​each HC and LC were identified. The HC and LC sequences are identified as follows: MWTx-001 (SEQ ID NO: 61 (HC) and 63 (LC)); MWTx-002 (SEQ ID NO: 65 (HC) and 67 (LC)); and MWTx-003 (SEQ ID NO: 69 (HC) and 71 (LC)). It will be understood that, based on the isolation and sequencing of the monoclonal antibodies from the hybridoma cell line producing the monoclonal antibody, the antibodies may be monoclonal antibodies isolated from the antibody-producing cell line, or recombinant monoclonal antibodies produced by recombinant expression of the known HC and LC of the antibody. The hybridoma cell line producing the MWTx-001 monoclonal antibody was deposited with the American Type Culture Collection under ATCC Accession No. PTA-126759 on May 27, 2020, under the terms of the Budapest Treaty. 10801 University Boulevard, Manassas, Virginia, 20110, USA. The hybridoma cell line producing the MWTx-002 monoclonal antibody was deposited with the American Type Culture Collection under ATCC Accession No. PTA-126760 on May 27, 2020, under the terms of the Budapest Treaty. 10801 University Boulevard, Manassas, Virginia, 20110, USA. The hybridoma cell line producing the MWTx-003 monoclonal antibody was deposited with the American Type Culture Collection under ATCC Accession No. PTA-126761 on May 27, 2020, under the terms of the Budapest Treaty. 10801 University Boulevard, Manassas, Virginia, 20110, United States.

[0096] b. Humanized variants

[0097] Humanized antibodies comprising CDRs derived from non-human sources grafted into human-derived antibody frameworks are expected to be non-immunogenic when administered to human subjects. As demonstrated by the exemplary embodiments disclosed in Example 2, humanized anti-TMPRSS6 antibody variants were successfully generated, tested, optimized, and selected. Multiple candidate HC and LC variants were developed, where each HC or LC variant had identical CDR sequences, but the variable region framework sequences could vary at more than 90% of the framework positions, and these variants were tested in different HC / LC combinations to identify combinations with desired characteristics. After preliminary design and testing, variants showing desired antigen-binding affinity were selected for further evaluation and development, including but not limited to modifying some parent CDR sequences to avoid potential unwanted events such as aspartate isomerization, and modifying some constant regions (Fc) to achieve desired functions such as minimizing antibody-dependent cellular cytotoxicity (ADCC), to obtain humanized variants hzMWTx-001Var (SEQ ID NO: 73 (HC) and 75 (LC)), hzMWTx-002Var (SEQ ID NO: 77 (HC) and 79 (LC)), and hzMWTx-003Var (SEQ ID NO: 81 (HC) and 83 (LC)).

[0098] c. Anti-TMPRSS6 antibodies that increase hepcidin promoter activity

[0099] As disclosed herein, antibodies for treating iron overload disorders characterized by decreased hepcidin expression may modulate the activity of at least one component involved in hepcidin expression, wherein the component may be the activity of the hepcidin promoter. As demonstrated by using the exemplary embodiment of 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 ( Figures 2A-2F ), whereas the isotype control at the same concentration did not increase HAMP promoter activity.

[0100] d. Anti-TMPRSS6 antibodies with high affinity for targets in relevant biological contexts

[0101] Anti-TMPRSS6 antibodies showed high affinity for a biologically appropriate target, namely human TMPRSS6 expressed on the cell surface. Figure 3M As demonstrated by the exemplary embodiments of affinity measurements using three different methods disclosed in , the monoclonal antibodies MWTx-001, MWTx-002 and MWTx-003, as well as the humanized variants hzMWTx-001Var, hzMWTx-002Var and hzMWTx-003Var consistently exhibit favorable affinity properties for therapeutically effective antibodies or antibody fragments.

[0102] e. Anti-TMPRSS6 Antibodies Cross-Reactive with Non-Human Targets

[0103] It is desirable that therapeutically useful antibodies or antibody fragments have sufficient cross-reactivity with non-human targets (non-human homologs) from sources relevant for further studies, such as preclinical efficacy studies, animal models of disease, toxicology studies, etc., such that the antibodies or antibody fragments should recognize, for example, mouse homologs and / or primate homologs, such as homologs from cynomolgus monkeys. As demonstrated by the exemplary embodiments 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.

[0104] f. Anti-TMPRSS6 antibodies specifically bind to TMPRSS6 (matriptase-2)

[0105] Antibodies with high levels of specific binding to target proteins and low cross-reactivity with homologous proteins in the same organism are expected to have reduced or no off-target effects. The anti-TMPRSS6 antibodies provided herein show 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 Figure 5A -R, monoclonal antibodies MWTx-001, MWTx-002 and MWTx-003, and their humanized variants hzMWTx-001Var, hzMWTx-002Var and hzMWTx-003Var showed specific binding to human TMPRSS6 (matriptase-2) and did not show detectable cross-reactivity with homologous human matriptase, i.e., these antibodies did not show detectable binding to matriptase-1 (ST14) or matriptase-3 (TMPRSS7).

[0106] g. Anti-TMPRSS6 Antibodies with Dose-Dependent Effects in Vivo on Hormones and Symptoms Associated with Iron Overload Disorders

[0107] Antibodies that can increase the level of serum hepcidin, a hormone that controls iron absorption and mobilization from iron stores, are expected to reduce, improve, or prevent the symptoms of iron overload disorders, particularly the symptoms of elevated serum iron levels. As demonstrated by the exemplary embodiments 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 an increase in serum hepcidin levels ( Figures 6A-6C ), reduction in serum iron levels ( Figures 6D to 6F ), and increased hepcidin RNA levels in the liver ( Figures 6G-6I These effects were dose-dependent, which can be interpreted as indicating (without wishing to be bound by the mechanism of action) that the dose-dependent in vivo effects of anti-TMPRSS6 antibodies indicate that a skilled artisan can determine an effective amount (dose) for a given subject.

[0108] h. Anti-TMPRSS6 Antibodies for the Treatment of β-Thalassemia

[0109] When administered to subjects (i.e., subjects known or suspected of having iron overload disorders) exhibiting animal models of the disease, antibodies and antibody fragments that can alleviate one or more symptoms of iron overload disorders in vivo are expected to have therapeutic effectiveness for clinical use. As demonstrated by the exemplary embodiments shown in Example 7 using a Th3 / + mouse model of beta-thalassemia, the administration of anti-TMPRSS6 monoclonal antibody MWTx-003 results in multiple effects, including but not limited to, reducing liver non-heme iron, increasing serum hepcidin, increasing liver hepcidin RNA, alleviating splenomegaly, increasing red blood cell count (RBC), increasing hematocrit (HCT), reducing red blood cell distribution width (RDW), and increasing the production of mature red blood cells (increasing erythropoiesis) compared to isotype controls. Each of these effects can be understood as an improvement in the symptoms of the disorder. Symptoms of the disorder are manifested in multiple biological systems, including, but not limited to, effects in the liver (effects on liver non-heme iron, liver hepcidin RNA), effects in the blood (effects on serum iron levels, circulating hormone levels, particularly serum hepcidin levels, RBCs, 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 precursor cell types and the abundance of mature erythrocytes in the erythropoietic site). Administration of anti-TMPRSS6 antibodies improves multiple symptoms across disease model subjects, shifting measured symptom levels away from levels seen in isotype controls (untreated disease) for the disease model and toward levels seen in wild-type littermates (which represent normal levels in genetically similar subjects who are not known or suspected of having the disease). Without wishing to be bound by theory or mechanism of action, it is understood that ineffective erythropoiesis is a driving force for aberrant hepcidin repression, leading to increased iron absorption and iron overload, such that treatments that improve differentiation and maturation of nucleated erythrocytes into erythrocytes should be therapeutically beneficial for treating iron overload disorders. Non-limiting exemplary embodiments herein disclose anti-TMPRSS6 antibody therapies that increase differentiation and maturation of nucleated erythrocytes into erythrocytes and also reduce iron loading.

[0110] i. Anti-TMPRSS6 antibodies for the treatment of polycythemia vera (PV)

[0111] Accordingly, certain aspects of the present disclosure provide methods and related compositions for treating conditions associated with dysregulated iron metabolism in a subject suffering from a myeloproliferative neoplasm (MPN) associated with JAK2 / STAT5 overactivation (e.g., polycythemia vera). Various aspects of the present disclosure relate to methods and compositions (e.g., anti-TMPRSS6 antibodies) that can be used to modulate iron metabolism for treating polycythemia vera in a subject with JAK2 / STAT5 overactivation.

[0112] Polycythemia vera (PV) is a chronic myeloproliferative neoplasm. PV is characterized by polycythemia, myeloid and megakaryocytic hyperplasia, fatigue, aquagenic pruritus, microvascular symptoms, and symptomatic splenomegaly. In some embodiments, overactivation of the JAK2 / STAT5 pathway leads to unregulated cell (e.g., hematopoietic progenitor cell) proliferation, thereby causing erythrocytosis, leukocytosis, and thrombocytosis in subjects with PV. Complications of polycythemia vera include an increased risk of arterial and venous thrombosis and the potential for progression to myelofibrosis (MF) and MPN-blast phase (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).

[0113] In some embodiments, a subject with polycythemia vera exhibits a phenotypic profile of polycythemia vera prior to treatment (e.g., a treatment described herein). In some embodiments, the subject has polycythemia vera relative to a subject without polycythemia vera. Polycythemia vera refers to the production of excess red blood cells. Polycythemia vera can be assessed by measuring hematocrit (HCT), hemoglobin, and bone marrow cell morphology in the subject (see, e.g., Mithoowani et al., Investigation and management of erythrocytosis, CMAJ. 2020 Aug 10;192(32):E913–E918).

[0114] In some embodiments, subjects with polycythemia vera associated with PV have an increased hematocrit (HCT) relative to subjects without PV. Hematocrit is the percentage of red blood cell volume in the blood. The normal range of HCT is about 38% to 48% for men and about 35% to 45% for women. In some embodiments, subjects with PV have polycythemia vera and exhibit increased HCT levels above 48% in women and above 52% in men. In some embodiments, subjects with polyps have an HCT level of greater than 48%, greater than 49%, greater than 50%, greater than 51%, greater than 52%, greater than 53%, greater than 54%, greater than 55%, greater than 56%, greater than 57%, greater than 58%, greater than 59%, greater than 60%, greater than 61%, greater than 62%, greater than 63%, greater than 64%, greater than 65%, greater than 66%, greater than 67%, greater than 68%, greater than 69%, greater than 70%, greater than 71%, greater than 72%, greater than 73%, greater than 74%, greater than 75%, or greater. In some embodiments, subjects with polyps with high HCT levels are at greater risk of developing thrombotic events (TE). In some embodiments, increased TE leads to more complications, such as cardiovascular complications, thereby increasing the morbidity and mortality of polyps. In some embodiments, cytoreductive therapy, phlebotomy, and / or apheresis may be used to reduce excess red blood cells to reduce HCT. HCT can be measured by any suitable method known in the art. Cytoreductive therapy refers to certain medications used to treat a decrease in the level of blood cells. Cytoreductive therapy includes interferon therapy, hydroxyurea, hydroxycarbamide, ruxolitinib and / or anagrelide.

[0115] In some embodiments, subjects with PV-associated polycythemia have increased hemoglobin (Hb) levels relative to subjects without PV. The normal range for Hb is about 13 g / dL to 17 g / dL for men and about 11 g / dL to 15 g / dL for women. In some embodiments, subjects with PV exhibit increased Hb levels above 16.5 g / dL in men and above 16 g / dL in women. In some embodiments, a subject with polymyalgia has an Hb level greater than 16 g / dL, greater than 16.2 g / dL, greater than 16.5 g / dL, greater than 16.8 g / dL, greater than 17 g / dL, greater than 17.2 g / dL, greater than 17.5 g / dL, greater than 17.8 g / dL, greater than 18 g / dL, greater than 18.2 g / dL, greater than 18.5 g / dL, greater than 18.8 g / dL, greater than 19 g / dL, greater than 19.2 g / dL, greater than 19.5 g / dL, greater than 19.8 g / dL, greater than 20 g / dL, or greater. Hemoglobin levels can be measured by any suitable method known in the art.

[0116] In some embodiments, a subject with PV-associated erythrocytosis exhibits trilineage hypercellularity in a bone marrow biopsy. In some embodiments, trilineage hypercellularity in the bone marrow is manifested as marked hyperplasia of erythroid, granulocyte, and megakaryocyte cells, with polymorphic mature megakaryocytes (subject (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, such as a bone marrow biopsy.

[0117] In some embodiments, a subject with PV-related erythrocytosis may have lower serum erythropoietin (EPO) levels (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 less). Serum EPO can be measured by any suitable method known in the art.

[0118] In some embodiments, the subject has an increased red blood cell distribution width (RDW) relative to a subject who does not have polycythemia vera. The red blood cell distribution width (RDW) test measures the difference in the volume and size of red blood cells (erythrocytes). In some embodiments, a high RDW is associated with a higher risk of thrombosis in polycythemia vera subjects (see, e.g., Liu et al., RBC distribution width predicts thrombosis risk in polycythemia vera, Leukemia, Vol. 36, pp. 566-568 (2022).

[0119] In some embodiments, the subject has leukocytosis relative to a subject who does not have polycythemia vera. Leukocytosis refers to a higher than normal white blood cell count. In some embodiments, a subject with polycythemia vera exhibits leukocytosis in the absence of other factors that may cause an increase in white blood cell count (e.g., infection, acute inflammation).

[0120] In some embodiments, subjects with polycythemia vera have an enlarged spleen. In polycythemia vera, the bone marrow produces too many red blood cells, white blood cells, and platelets, which can cause the spleen to become larger. In some embodiments, an enlarged spleen indicates disease progression in polycythemia vera (Lee, et al., Volumetric Splenomegaly in Patients With Polycythemia Vera, J Korean Med Sci. 2022 Mar 21;37(11):e87).

[0121] In some embodiments, PV patients are iron-deficient at the onset of disease and / or during the course of their disease. However, the coexistence of iron deficiency and erythrocytosis presents a physiological disconnect. In some embodiments, subjects with PV exhibit hepcidin suppression (i.e., lower hepcidin levels) compared to subjects who do not have PV. In some embodiments, hepcidin suppression in subjects with PV is due to increased erythropoietic activity. In some embodiments, increased erythropoiesis suppresses hepcidin via the erythroid-secreted 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 suppression leads to iron mobilization from hepatocyte stores, export after circulation through splenic macrophages, and increased iron absorption by duodenal enterocytes, which leads to recovery from iron deficiency. However, in some embodiments, in polycythemia vera subjects (e.g., polycythemia vera subjects with one or more JAK2 mutations described herein), hepcidin suppression leads to greater iron deficiency (see, e.g., Ginzburg et al., Dysregulated iron metabolism in polycythemia vera: etiology and consequences, Leukemia, Vol. 32, pp. 2105-2116 (2018)). In some embodiments, other conditions in polycythemia vera, such as inflammation, counteract hepcidin suppression. Abnormal hepcidin expression combined with iron deficiency suggests that dysregulated iron metabolism is an important component of the pathobiology of polycythemia vera. In some embodiments, subjects with polycythemia vera have decreased serum hepcidin relative to subjects without polycythemia vera. In some embodiments, subjects with polycythemia vera have decreased serum pro-hepcidin relative to subjects without polycythemia vera (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 pro-hepcidin can be measured by suitable methods known in the art, such as hepcidin prohormone ELISA, hepcidin ELISA, RT-PCR, and the like.Although the exact mechanism of hepcidin suppression and iron deficiency in PV patients remains elusive, previous studies have shown, in some embodiments, that therapeutic agents that increase hepcidin levels in subjects with PV (e.g., ruxolitinib, hepcidin mimetics) improve PV-related symptoms (e.g., reduction in hematocrit (HCT), reduction in 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).

[0122] In some embodiments, the development of polycythemia vera 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 polycythemia vera has a mutation in the JAK2 gene (e.g., a JAK2 mutation that causes overactivation 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 polycythemia vera has one or more JAK2 mutations in the pseudokinase domain of the JAK2 gene (e.g., exon 12 to exon 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 overactivation of JAK2, which leads to enhanced signaling through STAT5 (Kleppe 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, after EPO binds to EpoR, JAK2 mediates signaling through the erythropoietin receptor (EpoR). Activation of EpoR / JAK2 triggers multiple signaling pathways that regulate the survival, proliferation, and differentiation of erythroid precursors.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 polycythaemia 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, EPO-independent JAK2 / STAT5 activation results in continued growth and proliferation of cells carrying the mutation (e.g., hematopoietic progenitor cells). In some embodiments, EPO-independent JAK2 / STAT5 activation leads to polycythemia. 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 overactivation of JAK2. 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 the absence of exon 14 (see, e.g., Regimbeau et al., Genetic Background of Polycythemia Vera, Genes (Basel). 2022 April; 13(4): 637). In some embodiments, the subject has a 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 leads to overactivation of JAK2 / STAT5 signaling by destabilizing the inhibitory JH2-JH1 interface and / or promoting activation of JH1 (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 affects the severity of polymyalgia (PV). In some embodiments, the subject with PV is a heterozygote for the JAK2 V617F mutation. In some embodiments, the subject with PV is a homozygote for the JAK2 V617F mutation. In some embodiments, the JAK2 exon 14 mutation (e.g., the JAK2 V617F mutation) leads to EPO-independent JAK2 / STAT5 overactivation. In some embodiments, PV subjects with a JAK2 exon 14 mutation (e.g., a JAK2V617F mutation) have lower hepcidin levels compared to subjects who do not have PV. In some embodiments, subjects with a JAK2 exon 14 mutation may have one or more other mutations described herein.

[0123] In some embodiments, a subject with polycythemia vera has one or more JAK2 exon 12 mutations. In some embodiments, a subject with polycythemia vera does not have the JAK2 V617F mutation. In some embodiments, a subject with polycythemia vera who is negative for the JAK2 V617F mutation has one or more mutations in JAK2 exon 12. In some embodiments, the JAK2 exon 12 mutation leads to overactivation of the JAK2 / STAT5 pathway. In some embodiments, the JAK2 exon 12 mutation leads to polycythemia vera. In some embodiments, subjects with polycythemia vera who have a JAK2 exon 12 mutation have similar serum and liver iron levels compared to subjects with polycythemia vera who carry the JAK2 V617F mutation. In some embodiments, subjects with PV who have a JAK2 exon 12 mutation have higher hemoglobin levels, higher ERFE levels, and even lower hepcidin levels compared to subjects with PV who have a JAK2V617F 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 who have a JAK2 exon 12 mutation have a similar prognosis to subjects with a JAK2V617F 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 or more exon 12 mutations, including but not limited to F537-K539delinsL, N542-E543del mutation, H538QK539L, V536-I546 dup11, V536-F547 dup, F537-I546dup10F547L, F537IK539I, H538-K539delinsL, H538-K539del, H538DK539L1540S, H538G, K539L, K539E, I540-E543delinsMK, I540-E542delinsS, R541-E543delinsK, N542-E543del, D544-L545del, or S47insL1540-F547dup8. (See, e.g., Regimbeau et al., Genetic Background of Polycythemia Vera, Genes (Basel). 2022 Apr; 13(4): 637).

[0124] In some embodiments, the subject with polycythemia vera has one or more JAK2 exon 13 mutations. In some embodiments, the JAK2 exon 13 mutation results in overactivation of the JAK2 / STAT5 pathway. In some embodiments, the JAK2 exon 13 mutation results in polycythemia vera. In some embodiments, the subject with polycythemia vera has lower hepcidin levels than a subject without polycythemia vera. In some embodiments, the subject with polycythemia vera has 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).

[0125] In some embodiments, the subject with polycythemia vera 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 polycythemia vera. In some embodiments, the subject with polycythemia vera has lower hepcidin levels than a subject without polycythemia vera. In some embodiments, the subject with polycythemia vera has an 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).

[0126] In some embodiments, the subject with PV has a non-JAK2 mutation that results in JAK2 / STAT5 overactivation. For example, a subject with polycythemia vera may have a mutation in the lymphocyte adaptor protein (LNK) gene (also known as the 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 regulate signaling mediated by lineage-specific cytokines (e.g., TPO and EPO), thereby controlling megakaryocyte and erythroid development, respectively. In some embodiments, LNK negatively regulates EPO receptor (EpoR) signaling by inhibiting pathways in primary nucleated erythrocytes (e.g., the JAK2 / STAT5 pathway). In some embodiments, a subject with polycythemia vera has a mutation in the LNK gene that results in a loss of function of LNK, which then results in 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. Disorders. Am. J. Hematol. 2016; 91: 248–251). In some embodiments, a PV subject with an LNK mutation does not have a JAK2 mutation. In some embodiments, a PV subject with an LKN mutation also has a JAK2 mutation (e.g., a JAK2 V617F mutation).

[0127] In some embodiments, the subject with PV has one or more non-JAK2 mutations, including but not limited to 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, RUNX 1 gene, CUX1 gene, ETV6 gene, 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. In some embodiments, non-JAK2 mutations may be present in subjects with JAK2 mutations (e.g., JAK2 V617F or exon 12 mutations) (see, e.g., Regimbeau et al., Genetic Background of Polycythemia Vera, Genes (Basel). 2022 April; 13(4): 637).

[0128] In some embodiments, the mutation that causes JAK2 / STAT5 overactivation (e.g., any of the mutations described herein) is an acquired mutation. In some embodiments, the mutation that causes JAK2 / STAT5 overactivation (e.g., any of the mutations described herein) is a somatic mutation. In some embodiments, the subject with PV acquires a mutation that causes JAK2 / STAT5 overactivation in cells in the bone marrow (e.g., any of the mutations described herein). In some embodiments, the subject with PV acquires a mutation that causes JAK2 / STAT5 overactivation in hematopoietic progenitor cells (e.g., any of the mutations described herein). In some embodiments, the subject with PV acquires a mutation that causes CD34 + CD38 -JAK2 / STAT5 overactivation mutation in hematopoietic progenitor cells (e.g., any of the mutations described herein). In some embodiments, the subject with polycythemia vera acquires a mutation that causes JAK2 / STAT5 overactivation in myeloid progenitor cells (e.g., any of the mutations described herein). In some embodiments, the subject with polycythemia vera acquires a mutation that causes JAK2 / STAT5 overactivation in megakaryocyte-erythroid progenitor cells (e.g., any of the mutations described herein) (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 USA. 2006 Apr 18; 103(16): 6224–6229; Spivak, The polycythemia vera stem cell, Leukemia Suppl. 2014 Dec; 3(Suppl 1): S23–S24).

[0129] In some aspects, the present disclosure provides 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 described herein. In some aspects, the present disclosure provides a method for treating polycythemia vera (PV) in a subject having bone marrow comprising cells with JAK2 / STAT5 overactivation, the method comprising administering to the subject an effective amount of an anti-TMPRSS6 antibody described herein.

[0130] 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) in a subject with PV (e.g., a subject with PV associated with JAK2 / STAT5 overactivation) relative to a subject before receiving the anti-TMPRSS6 antibody or a subject with PV who did not receive the anti-TMPRSS6 antibody (e.g., a subject with PV associated with JAK2 / STAT5 overactivation). or 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). 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) in a subject with PV (e.g., a subject with PV associated with JAK2 / STAT5 overactivation) relative to a subject before receiving the anti-TMPRSS6 antibody or a subject with PV who did not receive the anti-TMPRSS6 antibody (e.g., a subject with PV associated with JAK2 / STAT5 overactivation). The increase in hepcidin in a subject with polycythemia vera (PV) (e.g., a subject with polycythemia vera associated with JAK2 / STAT5 overactivation) results in an increase in iron-restricted erythropoiesis (e.g., an increase in hepcidin in a subject with polycythemia vera (PV) (e.g., a subject with polycythemia vera associated with JAK2 / STAT5 overactivation)).

[0131] In some embodiments, administration of an anti-TMPRSS6 antibody results in a decrease in liver iron (e.g., a decrease 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%, at least 95%) in a subject with PV (e.g., a subject with PV associated with JAK2 / STAT5 overactivation) relative to the subject before receiving the anti-TMPRSS6 antibody or a subject with PV who did not receive the anti-TMPRSS6 antibody. Hepatic iron levels can be assessed by appropriate laboratory tests, such as magnetic resonance imaging (MRI) (e.g., Henninger et al., Practical guide to quantification of hepatic iron with MRI, EurRadiol. 2020; 30(1): 383–393), liver biopsy, etc.

[0132] In some embodiments, administration of an anti-TMPRSS6 antibody results in a decrease in circulating iron, such as serum iron, in a subject with PV (e.g., a subject with PV associated with JAK2 / STAT5 overactivation) relative to a subject before receiving the anti-TMPRSS6 antibody or a subject with PV who has not received the anti-TMPRSS6 antibody (e.g., a subject with PV associated with JAK2 / STAT5 overactivation) (e.g., a decrease 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%, at least 95%). Circulating iron levels can be assessed by appropriate laboratory tests, such as measuring serum iron, transferrin saturation (TSAT), or total iron binding capacity (TIBC).

[0133] In some embodiments, administration of an anti-TMPRSS6 antibody results in a decrease in hematocrit (HCT) (e.g., a decrease 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%, at least 95%) in a subject with PV (e.g., a subject with PV associated with JAK2 / STAT5 overactivation) relative to a subject before receiving the anti-TMPRSS6 antibody or a subject with PV who did not receive the anti-TMPRSS6 antibody. Circulating iron levels can be assessed by appropriate laboratory tests, such as magnetic resonance imaging (MRI) (e.g., Henninger et al., Practical guide to quantification of hepatic iron with MRI, EurRadiol. 2020; 30(1): 383–393), liver biopsy, etc.

[0134] In some embodiments, administration of an anti-TMPRSS6 antibody results in a decrease in red blood cell count in a subject with PV (e.g., a subject with PV associated with JAK2 / STAT5 overactivation) relative to a 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 overactivation) (e.g., a decrease 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%, at least 95%). Red blood cell count can be assessed by appropriate laboratory tests, such as blood smears, cell counters, flow cytometry, and the like.

[0135] 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 overactivation) relative to a 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 overactivation) (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%, at least 95%). RDW can be assessed by appropriate laboratory tests, such as blood tests.

[0136] In some embodiments, administration of an anti-TMPRSS6 antibody results in a decrease in erythroid progenitor cells (e.g., a decrease in erythroid progenitor cells (e.g., in the 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%, at least 95%) in a subject with PV (e.g., a subject with PV associated with JAK2 / STAT5 overactivation) relative to a subject before receiving the anti-TMPRSS6 antibody or a subject with PV but not receiving the anti-TMPRSS6 antibody. Erythroid progenitor cells can be assessed by appropriate laboratory tests, such as bone marrow biopsy, flow cytometry, and the like.

[0137] In some embodiments, administration of an anti-TMPRSS6 antibody results in a decrease in plasma hemoglobin levels in a subject with PV (e.g., a subject with PV associated with JAK2 / STAT5 overactivation) relative to a 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 overactivation) (e.g., a decrease 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%, at least 95%). Plasma hemoglobin levels can be assessed by appropriate laboratory tests, such as blood tests, etc.

[0138] 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 overactivation) relative to a 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 overactivation) (e.g., a decrease in MCV 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%, at least 95%). MCV can be assessed by appropriate laboratory tests, such as blood tests, etc.

[0139] 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 PV (e.g., a subject with PV associated with JAK2 / STAT5 overactivation) relative to a 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 overactivation) (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%, at least 95%). White blood cell counts can be assessed by appropriate laboratory tests, such as blood smears, cell counters, flow cytometry, and the like.

[0140] In some embodiments, administration of an anti-TMPRSS6 antibody results in a reduction in thrombotic events (TE) in a subject with PV (e.g., a subject with PV associated with JAK2 / STAT5 overactivation) relative to a 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 overactivation) (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%, at least 95%). TE can be determined by appropriate laboratory tests and clinical visits.

[0141] In some embodiments, administration of an anti-TMPRSS6 antibody results in a decrease in the frequency of phlebotomy in a subject with PV (e.g., a subject with PV associated with JAK2 / STAT5 overactivation) relative to a 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 overactivation) (e.g., a decrease 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%, at least 95%). The need for phlebotomy can be determined by a hematological assessment of the subject, such as testing for 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% (e.g., 70% to 95%, 75% to 90%, 80% to 85%, 85% to 90%) phlebotomy independence in treated subjects.

[0142] In some embodiments, administration of an anti-TMPRSS6 antibody results in a decrease in the frequency of cytoreductive therapy in a subject with PV (e.g., a subject with PV associated with JAK2 / STAT5 overactivation) relative to a 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 overactivation) (e.g., a decrease 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%, at least 95%). The need for cytoreductive therapy can be determined by a hematological assessment of the subject, such as testing HCT, hemoglobin, red blood cell count, bone marrow biopsy, etc.

[0143] In some embodiments, administration of an anti-TMPRSS6 antibody results in improved therapeutic efficacy (e.g., reduced HCT, hemoglobin, red blood cell count, improved iron deficiency, etc.) in a subject in need thereof who has PV but is resistant to or intolerant of phlebotomy and / or cytoreductive therapy.

[0144] In some embodiments, the anti-TMPRSS6 antibodies described herein can be administered to a subject in need thereof weekly, biweekly, three-weekly, monthly, two-monthly, or three-monthly. In some embodiments, anti-TMPRSS6 is administered via intravenous injection, subcutaneous injection, or intraperitoneal injection. In some embodiments, the anti-TMPRSS6 antibodies described herein can be administered in a clinic or self-administered by the subject (e.g., via subcutaneous injection).

[0145] In some embodiments, administration of an anti-TMPRSS6 antibody results in a decrease from baseline on the Myeloproliferative Neoplasms Symptom Assessment Form (MPN-SAF). The MPN-SAF is a tool for MPN subjects (e.g., PV patients) to track their symptoms and monitor how they feel over time. Symptoms to be monitored include, but are not limited to, fatigue, rapid fullness when eating (early satiety), abdominal discomfort, inactivity, difficulty concentrating, night sweats, itching (pruritus), bone pain, fever (above 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 treated with an anti-TMPRSS6 antibody are expected to have a decrease from baseline in MPN-SAF of greater than 50% (e.g., greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or 100%).

[0146] In some embodiments, administration of an anti-TMPRSS6 antibody has few or minimal adverse effects on the treated subject, e.g., no hematological adverse events such as thrombocytopenia or neutropenia, no anti-drug antibodies, etc.

[0147] In some embodiments, the present disclosure provides methods of 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 interferons (e.g., Ropeinterferon alpha-2b-njft (Besremi), pegylated interferon), JAK2 inhibitors (e.g., Ruxolitinib, XL019, Fidatinib (SAR302503), Molotinib), JAK1 inhibitors (e.g., Itatinib), hepcidin mimetics (e.g., Rusfitide (PTG-300)), lysine-specific demethylase inhibitors (e.g., Bomedemstat (IMG-7298), TMPRSS6 antagonists (e.g., Sapablursen (ISIS 702843), SLN124), anti-TfR1 antibodies (e.g., PPMX-T003), MDM2 inhibitors (e.g., edanuline (RG7388), KRT-232), tyrosine kinase inhibitors (e.g., dasatinib, erlotinib, Gleevec, lestaurinib (CEP-701)), HDAC inhibitors (e.g., gilvestrastat (ITF2357), MK-0683), PI3K inhibitors (e.g., erbulisam (TGR-1202), telomerase inhibitors (e.g., imetelstat), phlebotomy, low-dose aspirin, or hydroxyurea.

[0148] When administered to a subject exhibiting an animal model of the disease (i.e., a subject known or suspected of having a myeloproliferative disorder), antibodies and antibody fragments that can alleviate one or more symptoms of a myeloproliferative disorder in vivo are expected to have therapeutic efficacy for 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 decrease in circulating red blood cell (RBC) counts, and hemoglobin (HGB) concentrations indicating a reduction in polycythemia vera, as well as an increase in hepcidin levels, a decrease in serum iron concentration, and differential effects on the spleen and liver, wherein administration of the anti-TMPRSS6 recombinant monoclonal antibody MWTx-003 did not cause significant changes in liver iron content, but caused a significant increase in iron deposition in splenic macrophages. Certain effects can be understood as improvements in symptoms of the disorder. Symptoms of the disorder are manifested in multiple biological systems, including, but not limited to, effects in the liver, spleen, blood (particularly serum hepcidin levels, RBCs, HCT, polycythemia), and bone marrow. Administration of anti-TMPRSS6 antibodies improves multiple symptoms across disease model subjects, shifting measured symptom levels away from levels seen in isotype controls (untreated disease) for the disease model and toward levels seen in wild-type littermates (which represent normal levels in genetically similar subjects not known or suspected of having the disease). Non-limiting exemplary embodiments herein disclose anti-TMPRSS6 antibody therapies that increase hepcidin levels and reduce polycythemia in subjects with PV.

[0149] III. Composition

[0150] Compositions are provided that include a safe and effective amount of an 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, dextrose, water, glycerol, ethanol, excipients, stabilizers, preservatives, or combinations thereof. It should be understood that the pharmaceutical formulation should be compatible with the mode of administration.

[0151] The anti-TMPRSS6 antibodies disclosed herein can be administered by any suitable means, including but not limited to injection or parenteral infusion. Parenteral infusion can include intramuscular, intravenous, intraarterial, intraperitoneal, subcutaneous administration or parenteral delivery to the liver. The anti-TMPRSS6 antibodies disclosed herein can be formulated for introduction into liver tissue or the vascular system for local delivery to the target tissue. The anti-TMPRSS6 antibodies disclosed herein can be administered using a device, or as a reservoir or in a sustained-release formulation (e.g., a semipermeable matrix or microcapsule of a solid hydrophobic polymer containing the antibody) to allow slow and / or quantitative and / or local delivery. The anti-TMPRSS6 antibodies disclosed herein can be formulated and administered using a colloidal drug delivery system (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in a coarse emulsion.

[0152] IV. Methods

[0153] Provided are methods for treating iron metabolism disorders using an effective amount of an anti-TMPRSS6 antibody disclosed herein. Without wishing to be bound by a particular mechanism of action, the provided methods for targeting TMPRSS6 using an anti-TMPRSS6 antibody disclosed herein result in multiple downstream effects, particularly on components (molecules, systems, processes) involved in iron metabolism and erythropoiesis. Without wishing to be bound by a particular mechanism of action, provided are methods for treating iron metabolism disorders using an effective amount of an anti-TMPRSS6 antibody disclosed herein to modulate the activity of components involved in iron metabolism. In particular, provided are methods for treating iron overload disorders associated with excess iron accumulation in tissues and organs, including disorders associated with or characterized by ineffective erythropoiesis, which may include, but are not limited to, beta-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 iron overload conditions associated with low hepcidin levels, particularly conditions associated with suppression of hepcidin expression, including diseases or conditions in which abnormal suppression of hepcidin expression is involved, by administering anti-TMPRSS6 antibodies that increase hepcidin expression. Methods are also provided for treating myeloproliferative disorders. Methods are also provided for treating polycythemia vera (PV). Methods are also provided for treating polycythemia vera (PV) associated with insufficient hepcidin suppression.

[0154] As provided herein, methods for treating iron metabolism disorders comprise administering an effective amount of an anti-TMPRSS6 antibody disclosed herein to a subject in need thereof, wherein administration of the effective amount of the anti-TMPRSS6 antibody improves at least one biological effect (symptom) associated with the disorder. Methods for treating iron metabolism disorders associated with suppression of hepcidin levels are provided, wherein administration of an effective amount of an anti-TMPRSS6 antibody disclosed herein to a subject in need thereof results in at least one of: an increase in hepcidin promoter activity, an increase in hepcidin transcription, an increase in hepcidin RNA levels, and an increase in hepcidin levels, particularly serum hepcidin levels. Methods for treating subjects known or suspected of having an iron overload disorder are provided, wherein administration of an effective amount of an anti-TMPRSS6 antibody results in one or more biological effects, including but not limited to, reduced liver 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). Methods are provided for treating a subject known or suspected of having an iron overload disorder 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, reduced liver 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).

[0155] Provided are methods and compositions for treating iron metabolism disorders, particularly iron overload disorders, and even more particularly iron overload disorders characterized by ineffective erythropoiesis, wherein administration of an effective amount of an anti-TMPRSS6 antibody results in treatment or amelioration of more than one biological effect or symptom associated with the disorder. Without wishing to be bound by theory or mechanism of action, it is understood that ineffective erythropoiesis, characterized by apoptosis of erythroid precursor cells, results in fewer mature erythrocytes produced in the bone marrow, and is a driving force for abnormal hepcidin suppression leading to increased iron absorption and iron overload. In accordance with this understanding, treatments that improve the differentiation and maturation of nucleated erythrocytes into erythrocytes should be therapeutically beneficial for treating iron overload disorders. Anti-TMPRSS6 antibody therapy increases the effectiveness of nucleated erythrocyte differentiation and maturation into erythrocytes, reduces iron load, increases hepcidin expression, and the like, maximizing the therapeutic benefits of the methods and compositions using the anti-TMPRSS6 antibodies disclosed herein.

[0156] Methods and compositions 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), are provided, wherein administration of an effective amount of an anti-TMPRSS6 antibody results in treatment or amelioration of more than one biological effect or symptom associated with the disorder. Without wishing to be bound by theory or mechanism of action, given the extent of iron deficiency observed in PV patients, the observation of insufficient hepcidin suppression in PV patients should be understood to suggest that impaired or dysregulated iron metabolism is an important component of the pathobiology of PV, and in particular, insufficient suppression of hepcidin levels is a component of the pathobiology of PV. In accordance with this understanding, treatments that modulate hepcidin expression should be therapeutically beneficial for treating myeloproliferative neoplasms characterized by erythroid hyperplasia, particularly polycythemia vera (PV). The effectiveness of anti-TMPRSS6 antibody therapy to reduce polycythemia and normalize hematocrit (HCT) levels, as well as to increase hepcidin expression, particularly maximizes the therapeutic benefit of the methods and compositions using the anti-TMPRSS6 antibodies disclosed herein.

[0157] The following examples are provided to illustrate but not to limit the invention. Example

[0158] Example 1: Antibody Production and Identification of Antibodies Binding to TMPRSS6

[0159] The production of novel monoclonal antibodies against TMPRSS6 was performed using rodent in vivo immunization and hybridoma technology under contract to the LakePharma Discovery Immunology group (LakePharma, Inc. San Carlos, CA). DNA-based immunization via tail vein injection of hydrodynamic gene transfer was performed in B6; SJL mice (The Jackson Laboratories) using a mixture of pLEV113_huTMPRSS6 and pLEV113_moTMPRSS6-TCE plasmid DNA (cloned at LakePharma, Inc). Sufficient plasma titers, as determined by fluorescence-activated cell sorting (FACS), were obtained, triggering downstream antibody recovery and screening activities. Electrofusion using a NEPA GENE ECFG21 Super Electro Cell Fusion Generator (NepaGene Co., Ltd., Ichikawa-City, Chiba, Japan), was performed with pooled splenocytes from two immunized mice and a myeloma fusion partner. The fusion material was plated in a total of ten (10) 384-well plates in hypoxanthine-aminopterin-thymidine medium that specifically selects for hybridoma cells rather than unfused myeloma partner cells. Hybridoma supernatants were initially screened for HuTMPRSS6 reactivity by FACS measurement to detect supernatants that gave positive staining signals on HEK293T cells expressing TMPRSS6 (a plasmid encoding huTMPRSS6-(His)6 (SEQ ID NO: 97) was transfected into HEK293T cells to select for TMPRSS6-expressing HEK293T cells) and negative staining on the parental (HEK293T) cells at day 10 after fusion. Hybridoma supernatants that gave positive staining signals on HEK293 cells expressing TMPRSS6 but negative staining on the parental (HEK293T) cells were designated as "hits" for further screening. 192 hits were identified in the primary FACS screen, and 143 hits were confirmed in the secondary and tertiary FACS screens.

[0160] Example 2. Functional Screening of Anti-TMPRSS6 Antibodies; Identification, Generation, and Sequencing of Monoclonal Antibodies and Humanized Variants Thereof

[0161] HAMP-luciferase reporter gene assay

[0162] Hepcidin promoter-luciferase reporter gene 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 mouse Hamp promoter as originally disclosed). For the HAMP-luciferase reporter gene 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 into HepG2 cells (ATCC, HB-8065) ​​along with a construct encoding TMPRSS6. Transfected HepG2 cells expressing TMPRSS6 were pretreated with various concentrations of purified mAb diluted in starvation medium for approximately 3 hours, 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), followed by treatment with recombinant hBMP6 (R&D Systems) at a final concentration of 25-60 ng / ml to trigger BMP-SMAD-mediated signaling. Purified mouse IgG (Sigma-Aldrich) or human IgG1 (BioXcell) was used as a control. After overnight treatment with hBMP6, cells were lysed and luciferase substrate was added. Luminescence readings from firefly luciferase and Renilla luciferase were each recorded by measuring total luminescence. Activity was calculated as the ratio of firefly luciferase luminescence / Renilla luciferase luminescence (control). The results of these assays are shown in Figures 2A-2F middle.

[0163] In vitro functional screening

[0164] To screen for functionally active hybridomas, the HAMP-luciferase reporter gene assay described above was used to test all 143 HuTMPRSS6 binding hybridomas ("hits"). Supernatants from ten (10) of the 143 HuTMPRSS6 binding hybridomas increased HAMP promoter activity (data not shown) and were identified as "active clones" to undergo further testing. As described in Example 4 below, these ten (10) active clones were tested for cross-reactivity against the murine target MoTMPRSS6, and three (3) showed binding to both HuTMPRSS6 and MoTMPRSS6 as measured by FACS. These three cross-reactive clones were further plated in 192 wells of a 384-well plate at a density of 1 cell / well to generate monoclonal hybridoma clones. The resulting subclones that exhibited the desired functional activity and cross-reactivity against non-human targets, such as murine TMPRSS6 (moTMPRSS6) and / or cynomolgus monkey TMPRSS6 (cynoTMPRSS6), were identified as MWTx-001, MWTx-002, and MWTx-003.

[0165] Sequences of anti-TMPRSS6 antibodies MWTx-001, MWTx-002, and MWTx-003

[0166] 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) with a unique mouse IgG specific primer set to amplify the target variable region for sequencing. A unique heavy chain and a unique light chain were identified for each anti-TMPRSS6 antibody. The nucleotide sequence of each heavy chain and each light chain was determined. The amino acid sequence encoded by the nucleotide sequence was determined, and the CDR regions were identified using the Kabat numbering system. Table 1 presents the amino acid sequences of the heavy and light chain variable regions of each of MWTx-001, MWTx-002 and MWTx-003, 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.

[0167]

[0168]

[0169]

[0170]

[0171] Generation and screening of humanized anti-TMPRSS6 antibody variants

[0172] The humanization of parental antibody utilizes CDR to be transplanted on the human antibody framework to carry out.First, the homology modeling of the 3-dimensional structure of parental antibody is carried out to set up the structural model of parental antibody. Based on the removal of the VH-VL interface position of overall sequence identity, coupling, the CDR specification position of similar classification and potential N-glycosylation site, the amino acid sequence about the variable fragment framework is identified. Humanized antibody is designed by producing multiple hybridization sequence, and the multiple hybridization sequence merges the selected part of parental antibody sequence with people's framework sequence. The isotype that is selected as forming (format) humanized antibody is IgG1 for heavy chain and IgG1 κ for light chain. Using 3D model, these humanized sequences are systematically analyzed by visual inspection and computer modeling to separate the sequence that is most likely to retain antigen binding. The goal is to maximize the amount of the people's sequence in the final humanized antibody while retaining the original antibody specificity. The humanized variants of pairing humanized VH and VL are then expressed and purified for affinity analysis.

[0173] In one round of designing, generating and testing variants as part of affinity analysis, four VH variants were generated in which the VH-CDRs of the parent antibody MWTX-003 were in corresponding positions in four different human IgG1 derived frameworks (SEQ ID NOs: 89-92), and four VL (VK) variants were generated in which the VL-CDRs of the parent antibody MWTX-003 were in corresponding positions in four different human IgG1κ derived frameworks (SEQ ID NOs: 93-96). A total of sixteen (16) humanized variants representing each 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), and were found to have KD values ​​in the nanomolar range from 4.16E-07 ( to 1.09E-08).

[0174] Variants that exhibit the desired antigen binding affinity are selected for further evaluation and development.In some cases, the parent CDR sequence is modified to avoid potentially undesirable events, such as aspartate isomerization.

[0175] In order to silence antibody effector functions, particularly antibody-dependent cellular cytotoxicity (ADCC), key amino acid residues in the Fc region were identified for all humanized antibody variants and mutated (substituted). Available guidance on Fc mutations to achieve the goal of canceling ADCC is used to provide a reference for this paper's mutations, such as the removal of native Fc N-linked glycosylation sites in hIgG1 (N297A mutation), or the replacement of leucine at positions 234 and 235 of the lower hinge region of Fc (LALA double mutation), as described by (Tamm A, Schmidt RE. IgG binding sites on human Fcgamma 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 FcgammaR: current models. Immunol Lett. 2002 Jun 3;82(1-2):57-65. doi:10.1016 / s0165-2478(02)00019-6). In the variants herein, an N297A mutation was introduced into the Fc of hzMWTx-001Var and hzMWTx-002Var antibodies, and a LALA mutation was introduced into the Fc of hzMWTx-003Var antibodies to achieve the same goal of reducing or silencing ADCC (Table 3, SEQ ID NOs: 73, 77, 81).

[0176] After evaluation, 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.

[0177] Recombinant production of humanized anti-TMPRSS6 antibody variants

[0178] Expression constructs for humanized anti-TMPRSS6 antibody variants were engineered with 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 verified by sequencing. For recombinant antibody production, the expression construct was used for transient transfection using the ExpiCHO expression system (ThermoFisher) following the manufacturer's instructions. The expressed antibody was purified by protein A affinity chromatography. The yield of antibody production from transient transfection ranged from 50 mg to 300 mg per liter, with a purity of >95% and an endotoxin level of <1 EU / ml.

[0179] Sequences of humanized anti-TMPRSS6 antibody variants hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var

[0180] Humanized anti-TMPRSS6 antibody variants hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var were selected for further testing. The sequences of the variable regions of each variable region are shown in Table 2 below, where the identified CDRs are indicated by underlining, and the changes made in the humanized variant CDR sequences relative to the parent antibody are indicated and discussed.

[0181]

[0182]

[0183]

[0184]

[0185]

[0186] Table 3 shows the complete heavy and light chain protein sequences 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 show the locations of the mutations (alterations) introduced to reduce ADCC as described above.

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199] Dose-dependent effect of anti-TMPRSS6 antibody on HAMP promoter activity

[0200] Figures 2A-2F Results from testing MWTx-001, MWTx-002, MWTx-003 and their humanized variants hzMWTx-001Var, hzMWTx-002Var, hzMWTx-003Var, respectively, at the indicated concentrations using the HAMP-luciferase reporter assay described above are shown. Figure 2A )、MWTx-002( Figure 2B )、MWTx-003( Figure 2C ) and the humanized variant hzMWTx-001Var ( Figure 2D )、hzMWTx-002Var( Figure 2E )、hzMWTx-003Var( Figure 2F ) each increased HAMP promoter activity in a dose-dependent manner. 50 Calculated as 3 μg / ml ( Figure 2A ). About EC of MWTx-002 50 Calculated as 1 μg / ml ( Figure 2B ). About EC of MWTx-003 50 Calculated as 2 μg / ml ( Figure 2C ). About EC of hzMWTx-001Var 50Calculated as 0.8 μg / ml ( Figure 2D ). About EC of hzMWTx-002Var 50 Calculated as 0.3 μg / ml ( Figure 2E ). About EC of hzMWTx-003Var 50 Calculated as 0.3 μg / ml ( Figure 2F ).

[0201] Example 3. Binding affinity of anti-TMPRSS6 antibodies

[0202] 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 biofilm interferometry ( Figure 3G-3M ).

[0203] Anti-TMPRSS6 mAb binding affinity measurement using cell surface ELISA

[0204] HEK293T cells stably expressing human TMPRSS6 (generated by LakePharma Inc as described above; SEQ ID NO: 97) were fixed with 4% paraformaldehyde (PFA) and washed with dPBS (Dulbecco's phosphate-buffered saline, Corning Cellgro) before incubation with various concentrations of anti-TMPRSS6 antibodies 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, the cells were washed with BSA medium and then incubated with HRP-conjugated goat anti-mouse IgG (Invitrogen) as a secondary antibody. Finally, the cells were washed with dPBS to remove unbound antibodies and developed with ELISA liquid substrate (Sigma-Aldrich), followed by termination of the reaction by adding the same volume of 1 M H2SO4. The cells were then stained at OD 450nm The bound antibody was measured by absorbance at . Figures 3A-3C middle.

[0205] Anti-TMPRSS6 mAb binding affinity measurement using FACS

[0206] HEK293T cells stably expressing human TMPRSS6 were collected and blocked with dPBS + 3% BSA, followed by incubation with various concentrations of anti-TMPRSS6 antibodies diluted in dPBS + 3% BSA. Purified mouse IgG was used as a control. After incubation, the cells were washed with dPBS and then incubated with goat anti-mouse IgG conjugated to APC (Jackson ImmunoResearch Inc) as a secondary antibody. Finally, the cells were washed with dPBS to remove unbound antibodies, resuspended with dPBS + 1 mM EDTA, and then subjected to FACS analysis was performed using a Flow Cytometer (ACEA Biosciences, Inc., San Diego CA). Bound antibodies were determined by measuring the average APC intensity after excitation at 640 nm and measuring emission (fluorescence) at 675 nm. The results of these assays are shown in Figures 3D-3F middle.

[0207] Anti-TMPRSS6 antibody affinity and binding kinetics measurements using biomembrane interferometry

[0208] Biofilm interferometry is used to Affinity measurements and binding kinetics determination of anti-TMPRSS6 antibodies using the RED96e system (Sartorius AG). Prehydrated anti-mouse IgG Fc capture (AMC) biosensors (for MWTx-001, MWTx-002, and MWTx-003 anti-TMPRSS6 antibodies, Figures 3G-3I ) or anti-human IgG Fc capture (AHC) biosensor (for hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var anti-TMPRSS6 antibodies, Figures 3J-3L ) were first equilibrated in 1xKB (kinetic buffer, 1xPBS pH 7.4 + 0.02% Tween-20 + 0.1% BSA) for 120 seconds for the first baseline, followed by incubation with 10 mg / ml 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) were loaded onto the AMC or AHC biosensor for 240 seconds. Then, a second baseline signal was established for 120 seconds, followed by binding to various concentrations of human etco-TMPRSS6-FLAG (SEQ ID NO: 102) (generated in-house by fusing the extracellular domain of human TMPRSS6 to a FLAG tag at the C-terminus) for 240 seconds. Finally, the analyte was dissociated in 1xKB for 360 seconds. Data analysis was performed using Octet Data Analysis HT Software. KD, k on 、k off and R 2 Summarized in Figure 3M middle.

[0209] Example 4: Cross-reactivity: Binding of anti-TMPRSS6 antibodies to human TMPRSS6 and non-human TMPRSS6

[0210] Cross-reactivity determination by FACS

[0211] Selected anti-TMPRSS6 antibodies were tested to determine whether any of the antibodies could bind to TMPRSS6 from mouse and / or cynomolgus monkey. 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) (generated in-house) 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 and then incubated with anti-TMPRSS6 antibodies diluted in dPBS + 3% BSA. After incubation, cells were washed with dPBS and then incubated with goat anti-mouse IgG conjugated with AlexaFluor-488 (Invitrogen) as a secondary antibody. Finally, cells were washed with dPBS to remove unbound antibodies, resuspended with dPBS + 1 mM EDTA, and then subjected to FACS analysis was performed using a Flow Cytometer (ACEA Biosciences, Inc., San Diego CA). Bound antibodies were determined by excitation at 488 nm and measuring emission at 530 nm (FITC-A). The results of these assays are shown in histograms. Figures 4A-4I For MWTx-001( Figure 4D ) and MWTx-003( Figure 4F ) observed cross-reactivity with mouse TMPRSS6, while MWTx-002 ( Figure 4E ) showed no detectable cross-reactivity with mouse TMPRSS6. Figure 4G )、MWTx-002( Figure 4H ) and MWTx-003( Figure 4I ) observed cross-reactivity with cynomolgus monkey TMPRSS6.

[0212] Determination of cross-reactivity by cell surface ELISA

[0213] Stably expressing mouse TMPRSS6 (generated by LakePharma Inc. as described above) Figure 4J 、 4L , 4N, 4P, 4R, 4T) or cynomolgus monkey (generated in-house as described above, Figure 4K 、 4M HEK293T cells (40, 4Q, 4S, 4U) were fixed with methanol (100%) and washed with dPBS (Dulbecco's phosphate-buffered saline, Corning Cellgro), and then incubated with various concentrations of anti-TMPRSS6 antibodies and humanized variants thereof diluted in BSA medium (DMEM+1% Pen / Strep+10mM HEPES+1mg / ml BSA (Sigma-Aldrich)). Purified mouse IgG ( Figures 4J-4O ) or human IgG1 ( Figure 4P-4U ) was used as a control. After incubation, cells were washed with BSA medium and then incubated with goat anti-mouse IgG conjugated with HRP as a secondary antibody (Invitrogen, Figures 4J-4O ) or anti-human IgG (Millipore, Figure 4P-4U Finally, the cells were washed with dPBS to remove unbound antibodies and developed with ELISA liquid substrate (Sigma-Aldrich), followed by termination of the reaction by adding the same volume of 1MH2SO4 ELISA liquid substrate. 450nm The bound antibody was measured by absorbance at . Figures 4J-4U For MWTx-001( Figure 4J ) and MWTx-003( Figure 4N ) anti-TMPRSS6 antibody and its humanized variant hzMWTx-001Var( Figure 4P ) and hzMWTx-003Var( Figure 4T ) anti-TMPRSS6 antibodies observed cross-reactivity with mouse TMPRSS6, while MWTx-002 ( Figure 4L ) anti-TMPRSS6 antibody or its humanized variant hzMWTx-002Var( Figure 4R ) anti-TMPRSS6 antibodies did not show detectable cross-reactivity with mouse TMPRSS6. Figure 4K )、MWTx-002( Figure 4M ) and MWTx-003( Figure 4O ) anti-TMPRSS6 antibody and its humanized variant hzMWTx-001Var( Figure 4Q )、hzMWTx-002Var( Figure 4S ) and hzMWTx-003Var( Figure 4U ) Cross-reactivity of anti-TMPRSS6 antibodies with cynomolgus monkey TMPRSS6 was observed.

[0214] Example 5: Target specificity: Binding of anti-TMPRSS6 antibodies to cognate matriptases.

[0215] To determine whether anti-TMPRSS6 antibodies bind to cognate matriptases, HEK293T cells overexpressing matriptase (ST14) (SEQ ID NO: 100) were collected. Figure 5B 、 5E , 5H, 5K, 5N, 5Q), and HEK293T cells overexpressing matriptase-3 (TMPRSS7) (SEQ ID NO: 101) ( Figure 5C 、 5F , 5I, 5L, 5O, 5R) (generated in-house). Stably expressing human TMPRSS6 (matriptase-2) (SEQ ID NO: 97) (generated by LakePharma Inc as described above, Figure 5A 、 5D , 5G, 5J, 5M, 5P) HEK293T cells were used as positive controls, and HEK293T cells ( Figures 5A-5R) was 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 a concentration of approximately 1 μg / ml for 1 hour. After incubation, cells were washed with dPBS and incubated with goat anti-mouse IgG conjugated to AlexaFluor-488 (Invitrogen, Figures 5A-5I ) or goat anti-human IgG conjugated to allophycocyanin (APC) (Jackson Immuno Research, Figures 5J-5R Finally, the cells were washed with dPBS and resuspended with dPBS+1 mM EDTA and then subjected to FACS analysis was performed by FlowCytometer. The fluorescence intensity was determined by excitation at 488 nm and 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 ) to determine the bound antibodies. The results of these assays are shown in histograms. Figures 5A-5R All antibodies showed binding to human TMPRSS6 (matriptase-2) ( Figure 5A 、 5D , 5G, 5J, 5M, 5P), and none of the antibodies showed binding to the homologous matriptase ST14 ( Figure 5B 、 5E , 5H, 5K, 5N, 5Q) or TMPRSS7( Figure 5C 、 5F , 5I, 5L, 5O, 5R). MWTx-001 anti-TMPRSS6 antibody and its humanized variant hzMWTx-001Var anti-TMPRSS6 antibody showed binding to human TMPRSS6 ( Figure 5A 、 5J ) and did not show binding to matriptase(ST14)( Figure 5B 、 5K ) or matriptase-3 (TMPRSS7) ( Figure 5C 、 5L ) binding. MWTx-002 anti-TMPRSS6 antibody and its humanized variant hzMWTx-002Var anti-TMPRSS6 antibody showed binding to human TMPRSS6 (matriptase-2) ( Figure 5D 、 5M) and did not show binding to matriptase(ST14)( Figure 5E 、 5N ) or matriptase-3 (TMPRSS7) ( Figure 5F 、 5O ). MWTx-003 anti-TMPRSS6 antibody and its humanized variant hzMWTx-003Var anti-TMPRSS6 antibody showed binding to human TMPRSS6 (matriptase-2) ( Figure 5G 、 5P ) and did not show binding to matriptase(ST14)( Figure 5H 、 5Q ) or matriptase-3 (TMPRSS7) ( Figure 5I 、 5R ) combination.

[0216] Example 6. Treatment with anti-TMPRSS6 antibodies in a mouse pharmacodynamic model

[0217] To investigate the pharmacodynamic response of anti-TMPRSS6 antibodies in vivo, 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 ( Figure 6C 、 6F , 6I, 6L) were injected intraperitoneally into wild-type C57BL / 6J mice. Mouse IgG2b (BioXcell, Figures 6A-6B , 6D-6E, 6G-6H, 6J-6K) or human IgG1 (BioXcell, Figure 6C 、 6F , 6I, 6L) were used as isotype controls. 20 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. 44 hours after hydrodynamic injection, mice were euthanized and liver tissue and blood were collected. Liver RNA was purified by EZgene Total RNA Purification Plus from Biomiga (San Diego, CA) according to the manufacturer's instructions. Mouse serum was obtained by centrifuging whole blood at 1500 x g for 10 minutes.

[0218] Effect of treatment with anti-TMPRSS6 antibodies on serum iron

[0219] Serum iron was measured by an in-house developed chromogenic assay ( Figures 6A-6CBriefly, mouse serum or iron standards (31–500 μg / dL) were mixed with a mixed acid solution (0.6 M trichloroacetic acid, 0.4 M sodium thioglycolate, 1 M HCl) by vortexing for 30 seconds. The mixture was incubated at 37°C for 10 minutes, followed by centrifugation at 10,000 × g for 10 minutes, and then developed in a color development solution (1.5 M sodium acetate, 0.5 mM bathophenanthroline disulfonate). The OD values ​​were then calculated. 535nm The absorbance was read at 400 nm. Serum iron concentration was calculated using a linear iron standard curve. 10 mg / kg MWTx-003 anti-TMPRSS6 antibody ( Figures 6A-6B ) and its humanized variant hzMWTx-003Var anti-TMPRSS6 antibody ( Figure 6C ) treatment significantly reduced serum iron.

[0220] Effect of treatment with anti-TMPRSS6 antibodies on serum hepcidin

[0221] Serum hepcidin ( Figures 6D-6F ). Briefly, diluted mouse serum or hepcidin standards were mixed with hepcidin-biotin conjugate and then added to a plate coated with anti-mouse hepcidin antibody. Serum hepcidin or hepcidin standards competed with hepcidin-biotin conjugate for binding to the coated anti-hepcidin antibody. The bound hepcidin-biotin conjugate was detected with streptavidin-conjugated horseradish peroxidase (HRP) and developed with TMB, followed by a stop solution. The plate was then plated at OD 450nm The absorbance was read at 4°C. Data were analyzed using Graphpad Prism 8 using four-parameter logistic (4-PL) curve fitting and interpolated with serum hepcidin concentrations. Hydrodynamic delivery of GFP-TMPRSS6 significantly reduced serum hepcidin levels ( Figure 6D ), while 10 mg / kg MWTx-003 anti-TMPRSS6 antibody ( Figures 6D-6E ) and its humanized variant hzMWTx-003Var anti-TMPRSS6 antibody ( Figure 6F ) treatment reversed hepcidin repression and significantly increased serum hepcidin levels.

[0222] Effects of treatment with anti-TMPRSS6 antibodies on hepatic hepcidin RNA

[0223] Quantification of hepatic hepcidin RNA by real-time PCR ( Figures 6G-6IBriefly, cDNA was first synthesized from liver RNA using iScript Reverse Transcription Supermix (Bio-Rad) according to the manufacturer's instructions. Hepcidin transcripts were amplified using the specific primers listed below and PCR amplified using SsoAdvanced on a Bio-Rad CFX96 qPCR instrument according to the manufacturer's instructions. TM Universal Detection was performed with GFP-TMPRSS6 Green Supermix (Bio-Rad). Samples were analyzed in triplicate and results were normalized to β-actin RNA levels (measured by transcription, amplification with primers listed below, and quantification as described above). Hydrodynamic delivery of GFP-TMPRSS6 significantly reduced hepcidin RNA in the liver ( Figure 6G ). 10mg / kg MWTx-003 anti-TMPRSS6 antibody ( Figures 6G-6H ) and its humanized variant hzMWTx-003Var anti-TMPRSS6 antibody ( Figure 6I Treatment with 5-Hamp inhibitor (Hamp) reversed the repression 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 TGCGAT-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).

[0224] Serum concentrations of MWTx-003 anti-TMPRSS6 antibody or its humanized variant hzMWTx-003Var anti-TMPRSS6 antibody were quantified by an in-house developed cell surface ELISA (described above). Figures 6J-6L 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 background control). Bound MWTx-003 anti-TMPRSS6 antibody was detected with HRP-conjugated goat anti-mouse IgG, and bound hzMWTx-003Var anti-TMPRSS6 antibody was detected with HRP-conjugated goat anti-human IgG. Color development was performed with TMB, followed by stop solution. The cells were then incubated at OD 450nmAbsorbance was read at 4°C. Samples were analyzed in triplicate and results were normalized to HEK293T controls. Data were analyzed using Graphpad Prism 8 using a four-parameter logistic (4-PL) curve fit and serum anti-TMPRSS6 antibody concentrations were interpolated.

[0225] Example 7. In vivo efficacy of anti-TMPRSS6 antibodies using a β-thalassemia mouse model.

[0226] To investigate the in vivo efficacy of anti-TMPRSS6 antibodies, a β-thalassemia mouse model (B6.129P2-Hbb-b1) was selected. tm1Unc Hbb-b2 tm1Unc / J, JAX stock number: 002683, Jackson Laboratories, Bar Harbor ME), referred to herein as Th3 / + mice. Th3 / + mice and their wild-type (WT) littermates at 4-5 weeks of age were fed an iron-sufficient diet (Teklad TD.80394), and Th3 / + mice were treated with 10 mg / kg MWTx-003 anti-TMPRSS6 antibody or mouse IgG2b isotype control every three days for 4 weeks, while WT littermates were not treated. At the end of the treatment period, mice were euthanized, and spleen, liver, femur, and blood samples were collected. Liver total RNA was purified, and serum was collected as described above.

[0227] Effects on blood cell count, splenomegaly, serum iron, serum hepcidin, and liver hepcidin RNA

[0228] Complete blood count (CBC) was performed by VETSCAN HM5 automated hematology analyzer ( Figures 7A-7D MWTx-003 anti-TMPRSS6 antibody treatment significantly increased red blood cell counts (RBC, Figure 7A ) and hematocrit (HCT, Figure 7C ), and reduced red blood cell distribution width (RDW, Figure 7D ), but for hemoglobin (HGB, Figure 7B ) had no obvious effect.

[0229] Spleen weights were measured, and treatment with MWTx-003 anti-TMPRSS6 antibody significantly reduced splenomegaly in Th3 / + mice ( Figure 7E ).

[0230] Serum iron was measured as described above. Treatment with MWTx-003 anti-TMPRSS6 antibody significantly reduced serum iron ( Figure 7F A similar chromogenic assay was used to measure liver non-heme iron ( Figure 7GBriefly, the minced liver tissue was dried at 65°C overnight, followed by digestion with mixed acid (3M HCl, 10% trichloroacetic acid) at 65°C for 20 hours. The digestion supernatant was then collected for color development in a color development solution (1.5M sodium acetate, 0.5mM bathophenanthroline disulfonate). 535nm Absorbance was read at 400 nm. Treatment with MWTx-003 anti-TMPRSS6 antibody significantly reduced liver non-heme iron ( Figure 7G ).

[0231] Serum hepcidin was measured by Hepcidin-Murine Compete ELISA kit as described above. Treatment with MWTx-003 anti-TMPRSS6 antibody significantly increased serum hepcidin ( Figure 7H ).

[0232] Hepatic hepcidin RNA was quantified by real-time PCR as described above. Treatment with MWTx-003 anti-TMPRSS6 antibody significantly increased hepatic hepcidin RNA ( Figure 7I ).

[0233] Serum concentrations of MWTx-003 anti-TMPRSS6 antibodies were quantified by an in-house developed cell surface ELISA as described above ( Figure 7J ).

[0234] Effects on erythropoiesis

[0235] To investigate the effect of MWTx-003 anti-TMPRSS6 antibody on erythropoiesis in Th3 / + mice, bone marrow was harvested from femurs (see Figures 7K-7M ), and harvest splenocytes from the spleen (see Figures 7N-7P ) and analyzed. Harvested cells were blocked with rat anti-mouse CD16 / CD32 antibodies (BD Biosciences) for 15 minutes, followed by staining with rat anti-mouse TER119 conjugated to FITC (BD Biosciences) and rat anti-mouse CD44 conjugated to APC (Invitrogen) on ice for 30 minutes. Washed cells were stained with the viability marker 7-AAD (BD Biosciences) on ice for 10 minutes, followed by staining with FACS analysis by flow cytometer. Select Ter119 + 7-ADD - Cells were isolated and density plots of cell size (FSC-H) were made using anti-mouse CD44. The plots were analyzed to identify cell types (cell clusters) and determine the abundance of each type (cluster). Figures 7K-7PRepresentative images in Figure 2 show that four distinct cell clusters corresponding to the successive stages of erythroid differentiation are distinguished from top to bottom and identified as: basophilic erythroblasts (cluster I), polychromatic erythroblasts (cluster II), orthochromatic erythroblasts and anucleated reticulocytes (cluster III), and mature erythrocytes (cluster IV). Figures 7K-7P As shown in , the percentage (%) value of each cluster in the sample was calculated as a measure of the abundance of the cell type in the cluster. The % value for each cell cluster (I), (II), (III), (IV) was calculated for each sample (bone marrow, spleen) from each animal in each treatment process as follows: WT (no treatment) 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 then the average value was calculated. On average, the population of basophilic erythroblasts (I) in bone marrow cells after four weeks showed a shift from 7.58% (Th3+w / MoIgG2b) to 6.52% (Th3+w / MWTx-003) (7.96% for WT), polychromatic erythroblasts (II) showed a shift from 54.20% (Th3+w / MoIgG2b) to 40.01% (Th3+w / MWTx-003) (28.53% for WT), orthochromatic erythroblasts and anucleated reticulocytes (III) showed a shift from 24.06% (Th3+w / MoIgG2b) to 29.73% (Th3+w / MWTx-003) (26.67% for WT), and mature red blood cells (IV) showed a shift from 4.54% (Th3+w / MoIgG2b) to 16.44% (27.66% for WT). On average, the basophilic erythroblastic population in the spleen after four weeks showed a shift from 0.71% (Th3 + w / MoIgG2b) to 0.91% (Th3 + w / MWTx-003) (0.46% for WT), and the polychromatic erythroblastic population (II) showed a shift from 45.76% (Th3 + w / MoIgG2b) to 19.25% (Th3 + w / MWTx-003) (12.23% for WT). %), orthochromatic erythroblasts and anucleated reticulocytes (III) showed a shift of 31.16% (Th3 + w / MoIgG2b) to 28.72% (Th3 + w / MWTx-003) (8.67% for WT), and mature erythrocytes (IV) showed a shift of 14.13% (Th3 + w / MoIgG2b) to 44.38% (Th3 + w / MWTx-003) (72.17% for WT). These results are shown in bar graphs, with respect to bone marrow shown in Figure 7Qand regarding the spleen is shown in Figure 7R middle.

[0236] In Th3 / + mice, treatment with the MWTx-003 anti-TMPRSS6 antibody ameliorated ineffective erythropoiesis, with a significant fraction of nucleated erythrocytes differentiating and maturing into red blood cells.

[0237] Example 8. Anti-TMPRSS6 Antibody Epitope Grouping

[0238] RED96e for MWTx-001( Figure 8A )、MWTx-002( Figure 8B ) and MWTx-003( Figure 8C ) Epitope grouping of anti-TMPRSS6 antibodies. First, etco-TMPRSS6-FLAG (as described above) was labeled with biotin using a biotinylation kit (Abcam). Prehydrated streptavidin (SA) biosensors were equilibrated in 1x KB buffer (as described above) for 60 seconds for the first baseline, followed by loading the SA biosensor with 10 mg / ml of biotinylated etco-TMPRSS6-FLAG for 300 seconds. Then, a second baseline signal was established for 60 seconds, followed by the antibody (MWTx-001, Figure 8A ;MWTx-002, Figure 8B ;MWTx-003, Figure 8C ) saturated for 600 seconds. Finally, a third baseline signal was established for 60 seconds, followed by competition with 50 μg / ml of MWTx-001, MWTx-002, or MWTx-003 in 1x KB for 300 seconds. The binding of MWTx-001 anti-TMPRSS6 antibody to etco-TMPRSS6-FLAG did not compete with MWTx-002 anti-TMPRSS6 antibody or MWTx-003 anti-TMPRSS6 antibody ( Figure 8A The binding of MWTx-002 anti-TMPRSS6 antibody to etco-TMPRSS6-FLAG did not compete with MWTx-001 anti-TMPRSS6 antibody, but competed with MWTx-003 anti-TMPRSS6 antibody ( Figure 8B The binding of MWTx-003 anti-TMPRSS6 antibody to etco-TMPRSS6-FLAG did not compete with MWTx-001 anti-TMPRSS6 antibody, but competed with MWTx-002 anti-TMPRSS6 antibody ( Figure 8C Data analysis was performed using Octet Data Analysis HT Software. Figure 8Dmiddle.

[0239] Example 9. Efficacy Study of Anti-TMPRSS6 Monoclonal Antibodies in Polycythemia Vera Mouse Model

[0240] The effect of anti-TMPRSS6 recombinant monoclonal antibody treatment on reversing polycythemia and normalizing hematocrit levels in a mouse model of polycythemia vera (PV) was evaluated.

[0241] B6N.129S6(SJL)-Jak2 tm1.1Ble / AmlyJ mice (JAX#031658), commonly known as Jak2 V617F-Fl / + , is a floxed strain with an inversion V617F mutation, carrying exon 14 downstream of the endogenous exon 14 of the Janus kinase 2 (Jak2) gene. The V617F mutation is commonly found in patients with myeloproliferative neoplasms and is present in approximately 95% of patients with polymyelocytic leukemia (PV). When mated with mice expressing tissue-specific Cre recombinase, the resulting offspring will have the floxed endogenous exon 14 removed and the V617F mutant exon 14 placed in the correct transcriptional orientation.

[0242] B6.Cg-Commd10 Tg(Vav1-icre)A2Kio / J mice (JAX#008610), commonly referred to as Vav-iCre, express an optimized variant of Cre recombinase (iCre) specifically in hematopoietic cells and can be used to generate conditional mutations in the hematopoietic progenitor compartment. V617F-Fl / + Offspring of the mice develop PV characterized by polycythemia and elevated hematocrit levels, and the phenotype can be reproduced by transplanting bone marrow cells from doubly transgenic mice into lethally irradiated wild-type recipient mice.

[0243] The recombinant mouse anti-TMPRSS6 monoclonal antibody MWTx-003 was designated r4K12B in this study, wherein the antibody is a recombinantly expressed form of the mouse monoclonal MWTx-003 and is Figures 9A-9HThe recombinant monoclonal antibody, which may be referred to as recombinant monoclonal antibody MWTx-003, recombinant MWTx-003, or MWT-003, is used in this in vivo repeat-dose study in a mouse model of polymyxin B (PV) to avoid potential immunogenicity and the development of anti-drug antibodies (ADA). The recombinant mouse anti-TMPRSS6 monoclonal antibody, r4K12B (the mouse counterpart of the humanized antibody hzMWTx-003Var), has a HC of SEQ ID NO: 69 (the amino acid sequence of the HC of mouse monoclonal MWTx-003) and a LC of SEQ ID NO: 71 (the amino acid sequence of the LC of mouse monoclonal MWTx-003) expressed from a vector in which nucleotides of SEQ ID NO: 70 (the HC coding sequence of MWTx-003) and nucleotides of SEQ ID NO: 72 (the LC coding sequence of MWTx-003) are inserted into a single vector with an engineered IRES between the HC and LC coding sequences, and the expressed polypeptide is purified.

[0244] Material

[0245] The following materials were used to evaluate the effect of anti-TMPRSS6 antibody treatment on reversing polycythemia and normalizing hematocrit levels in a mouse model of polycythemia vera.

[0246] r4K12B, a recombinant mouse monoclonal antibody produced in-house (recombinant MWTx-003)

[0247] a. Isotype: mouse IgG2b, κ

[0248] b.Batch number: LN211201

[0249] c. Concentration: 3.8 mg / mL in PBS, pH 7.4

[0250] d. Purity: >95% as determined by SDS-PAGE

[0251] e. Endotoxin: 0.71EU / mg

[0252] InVivoPlus Mouse IgG2b Isotype Control, purchased from BioXCell (#BP0086)

[0253] f. Clone: ​​MPC-11

[0254] g.Batch number: 779420O1

[0255] h. Concentration: 10.26 mg / mL in PBS, pH 7.0

[0256] i. Purity: >95% as determined by SDS-PAGE

[0257] j. Endotoxin: <1EU / mg

[0258] method

[0259] Animal studies

[0260] Wild-type C57BL / 6J (JAX#000664) male mice aged 10-12 weeks were purchased from The Jackson Laboratory and allowed to acclimate to the housing environment before the start of the study. All mice received a lethal dose of 1000 cGy whole-body irradiation at 3.45 Gy / min. 24 hours later, the Jak2 V617 / + 5×10 isolated from Vav-iCre double transgenic mice (both male and female mice were used) 6 Bone marrow cells were injected into each lethally irradiated recipient C57BL / 6J mouse via the lateral tail vein. Immediately after bone marrow transplantation (BMT), antibiotics (sulfamethoxazole and trimethoprim) were administered ad libitum in acidic drinking water (pH 2.5–3.0) for two weeks. BMT animals were monitored for the development of the polymyalgia (PV) phenotype by complete blood count using an automated hematology analyzer. Four weeks after BMT, when the PV phenotype was fully established, mice received intraperitoneal injections of the anti-TMPRSS6 antibody r4K12B (recombinant MWTx-003) or a mouse IgG2b isotype control antibody every four days for a total of three weeks. Four days after the last dose, animals were euthanized, and bone marrow, spleen, liver, and whole blood were harvested for analysis. The effects of anti-TMPRSS6 antibody treatment on erythroid profile, hematological parameters (including mean corpuscular volume (MCV) and mean RBC size), splenomegaly, and tissue iron accumulation were assessed.

[0261] Serum hepcidin, iron concentration, and tissue iron deposition

[0262] As described above, according to the manufacturer's instructions, Hepcidin-Murine Compete TM Serum hepcidin was measured by ELISA (Intrinsic Lifesciences, SKU#HMC-001). The results are shown in ( Figure 9E )middle.

[0263] Serum iron was measured using a chromogenic assay as described above.

[0264] Iron deposition in the spleen and liver was assessed by Perls' Prussian blue staining on 10% formaldehyde-fixed liver and spleen sections. Sectioning, staining, and imaging were contracted by Reveal Biosciences (San Diego, California). Figure 9H )

[0265] Analysis of hematological parameters

[0266] Red blood cell indices were analyzed by complete blood count (CBC) on an HM5 VetScan Hematology Analyzer. Figures 9A-9C )

[0267] Differentiation of nucleated erythrocytes was assessed in the bone marrow and spleen, respectively. Bone marrow harvested from the femur and splenocytes harvested from the spleen were analyzed by FACS as described above. The results are shown in ( Figure 9G )middle.

[0268] Measurement of anti-TMPRSS6 antibody concentration in mouse serum

[0269] Serum concentrations of r4K12B anti-TMPRSS6 antibody (recombinant MWTx-003) were quantified by an in-house developed cell surface ELISA as described above. The results are shown in ( Figure 9F )middle.

[0270] Statistical analysis

[0271] One-way ANOVA was used to compare three or more data sets using GraphPad Prism software. P < 0.05 was considered statistically significant.

[0272] result

[0273] Animal group assignment

[0274] During the acclimation process, the body weights of bone marrow recipient C57BL / 6J mice (all male) were measured for randomization to obtain similar mean body weights among the groups. Group allocation was performed according to the following table (Table 4).

[0275] Table 4. Experimental group assignments.

[0276]

[0277] Accept Jak2 V617 / + Development of the PV phenotype in mice with Vav-iCre bone marrow cells

[0278] Blood samples were collected from recipient mice (wild-type C57BL / 6J mice that received bone marrow transplantation (BMT) of Jak2V617 / +Vav-iCre bone marrow cells) and hematological parameters were analyzed 3 and 4 weeks after BMT. V617 / + In Vav-iCre double transgenic mice (designated the "PV reference strain"), the PV phenotype developed in recipient mice 3 weeks after BMT and was fully established by 4 weeks after BMT (Table 5).

[0279] Table 5. Hematological parameters in lethally irradiated C57BL / 6J recipients after BMT.

[0280]

[0281] Administration of anti-TMPRSS6 antibodies reverses Jak2 expression in PV V617 / + Reducing polycythemia and normalizing hematocrit levels in a mouse model

[0282] Four weeks after BMT, when the PV phenotype was fully established, mice (designated "PV phenotype" mice) received intraperitoneal injections of the anti-TMPRSS6 antibody r4K12B (recombinant MWTx-003) at dose levels of 2 mg / kg, 5 mg / kg, and 10 mg / kg, or 10 mg / kg mouse IgG2b isotype control every four days for three weeks. Endpoint analysis was performed four days after the last injection.

[0283] Following 2 weeks of treatment with the anti-TMPRSS6 antibody r4K12B, a dose-dependent trend toward reduction in hematocrit (HCT) levels, red blood cell (RBC) counts, and hemoglobin (HGB) concentrations was observed in mice receiving r4K12B compared to animals treated with an isotype control antibody (Table 6).

[0284] Table 6. Hematological parameters in mice receiving anti-TMPRSS6 antibodies for 2 weeks.

[0285]

[0286]

[0287] Results are expressed as mean ± SD, using one-way ANOVA with Dunnett's multiple comparison adjustment, ***P < 0.001, **P < 0.01, *P < 0.05 compared to the mIgG2b isotype control. N = 6 for the mIgG2b group, N = 8 for the 10 mg / kg group, and N = 7 for both the 5 mg / kg and 2 mg / kg groups.

[0288] Results after 3 weeks of treatment

[0289] Figures 9A-9C Hematological parameters HCT ( Figure 9A )、RBC( Figure 9B ) and HGB( Figure 9C ) endpoint measurement. Figures 9D-9E Endpoint measures for each treatment and dose level are also shown, where Figure 9DSplenomegaly (Splenomegaly Index measured as mg / g body weight) is shown. Figure 9E Serum hepcidin levels (ng / ml) are shown, while Figure 9F Shown are serum anti-TMPRSS6 r4K12B concentrations (μg / ml) measured by cell surface ELISA. Figure 9G Shown are FACS results measuring early erythroid precursors (cluster I, basophilic erythroblasts and cluster II, polychromatic erythroblasts) in the bone marrow (upper row) and spleen (lower row), showing results for WT (left panel, upper and lower), MoIgG2b isotype control (middle panel, upper and lower), and treatment with 10 mg / kg of anti-TMPRSS6 r4K12B (MWTx-003) (right panel, upper and lower). Figure 9H Shown are sections of liver (left) and spleen (right) stained to show iron content. Figures 9A-9H In the figures, the label MWTx-003 indicates treatment or measurement with the antibody r4K12B.

[0290] At the end of the 3-week treatment period, HCT levels in all r4k12B-treated groups were further reduced in a dose-dependent manner to levels similar to or lower than those seen in wild-type (WT) untreated animals ( Figure 9A Circulating RBC numbers and HGB concentrations were also reduced, with a significant reduction in polycythemia noted for the 10 mg / kg dose group ( Figure 9B -C). Splenomegaly was also observed in the 10 mg / kg dose group ( Figure 9D ) and expansion of early erythroid progenitors (i.e., cluster I, basophilic erythroblasts and cluster II, polychromatic erythroblasts) ( Figure 9G ), indicating the development of iron-restricted erythropoiesis. As expected, serum hepcidin increased significantly and persisted during treatment ( Figure 9E ), resulting in a dramatic reduction in serum iron concentrations below detection by colorimetric assays (data not shown). These observations indicate that, although the anti-TMPRSS6 antibody r4K12B (MWTx-003) is potent in reducing erythrocytosis and improving the PV phenotype, the dose and duration of treatment should be titrated to minimize the negative effects of erythrocyte iron deficiency. Figure 9GShown are representative FACS results measuring early erythroid precursors in the bone marrow (upper row) and spleen (lower row), where cluster I shows basophilic erythroblasts and cluster II shows polychromatic erythroblasts, showing results for WT (left panel, upper and lower), MoIgG2b isotype control (middle panel, upper and lower), and treatment with 10 mg / kg of anti-TMPRSS6 r4K12B (MWTx-003) (right panel, upper and lower). The total percentage of cluster I and cluster 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 total % of clusters I and II in the r4K12B group was statistically different from that in the moIgG2b group and wild-type control mice (P = 0.0399 and P = 0.0277, respectively), while there was no statistical difference between the wild-type and moIgG2b-treated groups.

[0291] Figure 9H Shown are Perls' Prussian blue staining of formalin-fixed liver sections (left panel) and spleen sections (right panel) from control animals treated with mouse IgG2b isotype control MoIgG2b (upper row) and animals treated with increasing doses of anti-TMPRSS6 r4K12B (labeled MWTx-003) as indicated to measure iron deposition. Figure 9H The results in demonstrated that administration of the anti-TMPRSS6 antibody r4K12B (MWTx-003) did not cause major changes in liver iron content, but caused a significant increase in iron deposition in splenic macrophages, with the increase being observed in a dose-dependent manner.

[0292] in conclusion

[0293] Subchronic treatment with anti-TMPRSS6 antibodies substantially alleviated polycythemia in a mouse model of polycythemia vera by limiting iron availability to erythroid precursors and normalized hematocrit levels. Anti-TMRSS6 antibody treatment offers a promising therapeutic approach in the management of polycythemia vera, in which polycythemia and high hematocrit levels are associated with a poor prognosis.

Claims

1. A method for treating polycythemia vera (PV) in a subject, wherein the PV is associated with overactivation of the JAK2 / STAT5 pathway, comprising administering to the subject an effective amount of an anti-TMPRSS6 antibody, wherein the antibody comprises: (a) a heavy chain complementary 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 complementary 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) an HC CDR1 comprising the amino acid sequence of SEQ ID NO: 2, an HC CDR2 comprising the amino acid sequence of SEQ ID NO: 3, an HC CDR3 comprising the amino acid sequence of SEQ ID NO: 4, an LC CDR1 comprising the amino acid sequence of SEQ ID NO: 7, an LC CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and an LC CDR3 comprising the amino acid sequence of SEQ ID NO: 9; (c) 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; (d) 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; (e) an 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) HC CDR1 comprising the amino acid sequence of SEQ ID NO:42, HC CDR2 comprising the amino acid sequence of SEQ ID NO:43, HC CDR3 comprising the amino acid sequence of SEQ ID NO:44, LC CDR1 comprising the amino acid sequence of SEQ ID NO:47, LC CDR2 comprising the amino acid sequence of SEQ ID NO:48, and LC CDR3 comprising the amino acid sequence of SEQ ID NO:

49.

2. A method for treating polycythemia vera (PV) in a subject having a bone marrow comprising cells with JAK2 / STAT5 overactivation, the method comprising administering to the subject an effective amount of an anti-TMPRSS6 antibody, wherein the antibody comprises: (a) a heavy chain complementary 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 complementary 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) an HC CDR1 comprising the amino acid sequence of SEQ ID NO: 2, an HC CDR2 comprising the amino acid sequence of SEQ ID NO: 3, an HC CDR3 comprising the amino acid sequence of SEQ ID NO: 4, an LC CDR1 comprising the amino acid sequence of SEQ ID NO: 7, an LC CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and an LC CDR3 comprising the amino acid sequence of SEQ ID NO: 9; (c) 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; (d) 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; (e) an 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) HC CDR1 comprising the amino acid sequence of SEQ ID NO:42, HC CDR2 comprising the amino acid sequence of SEQ ID NO:43, HC CDR3 comprising the amino acid sequence of SEQ ID NO:44, LC CDR1 comprising the amino acid sequence of SEQ ID NO:47, LC CDR2 comprising the amino acid sequence of SEQ ID NO:48, and LC CDR3 comprising the amino acid sequence of SEQ ID NO:

49.

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 1-3, wherein the subject has a JAK2 mutation. The method according to claim 4 , wherein the JAK2 mutation is a JAK2 gene exon 14 mutation.

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

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

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 1-8, wherein the JAK2 mutation is a JAK2 gene exon 12 mutation.

10. The method of claim 9, wherein 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 S547insLI540-F547dup8.

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

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

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

14. The method of claim 13, wherein the subject has a mutation in the following: 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 gene, CUX1 gene, ETV6 gene, 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.

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

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

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

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 16-18, wherein the one or more mutations occur in a megakaryocyte-erythroid progenitor cell.

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

21. The method of any one of claims 1-20, wherein the subject has an increased hematocrit (HCT) relative to a subject not suffering from PV.

22. The method of any one of claims 1-21, wherein the subject suffers from splenomegaly prior to administration.

23. The method of any one of claims 1-22, wherein the subject suffers from polycythemia prior to administration.

24. The method of any one of claims 1-23, wherein the subject suffers from leukocytosis prior to administration.

25. The method of any one of claims 1-24, wherein the subject suffers from thrombocythemia prior to administration.

26. The method of any one of claims 1-25, wherein the subject has increased hemoglobin relative to a subject not suffering from PV prior to administration.

27. The method of any one of claims 1-26, wherein the subject has increased red blood cell distribution width (RDW) relative to a subject not suffering from PV prior to administration.

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

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

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

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

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

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

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

35. The method of any one of claims 1-34, wherein administration of the antibody reduces early erythroid progenitor cells.

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

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

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

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

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

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

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

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

44. The method of claim 43, wherein the additional therapeutic agent for treating PV comprises an interferon (e.g., Ropeinterferon alpha-2b-njft (Besremi), pegylated interferon), a JAK2 inhibitor (e.g., Ruxolitinib, XL019, Fidatinib (SAR302503), Molotinib), a JAK1 inhibitor (e.g., Itatinib), a hepcidin mimetic (e.g., Rusfitide (PTG-300)), a lysine-specific demethylase inhibitor (e.g., Bomedemstat (IMG-7298), a TMPRSS6 antagonist (e.g., Sapablursen (ISIS 702843), SLN124), anti-TfR1 antibodies (e.g., PPMX-T003), MDM2 inhibitors (e.g., edanuline (RG7388), KRT-232), tyrosine kinase inhibitors (e.g., dasatinib, erlotinib, Gleevec, lestaurinib (CEP-701)), HDAC inhibitors (e.g., gilvestrastat (ITF2357), MK-0683), PI3K inhibitors (e.g., erbulisam (TGR-1202), telomerase inhibitors (e.g., imetelstat), phlebotomy, low-dose aspirin, or hydroxyurea.

45. The method of any one of claims 1-42, wherein the antibody comprises a heavy chain complementary 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 complementary 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.

46. ​​The method of any one of claims 1-43, wherein the antibody comprises: (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) VH comprising the amino acid sequence of SEQ ID NO: 41, and VL comprising the amino acid sequence of SEQ ID NO:

46.

47. The method of any one of claims 1-44, 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.

48. The method of any one of claims 1-45, wherein the antibody comprises: (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.

49. The method of any one of claims 1-46, 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.

50. The method of any one of claims 1-47, wherein the antibody comprises a heavy chain complementary 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 complementary 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.

51. The method of any one of claims 1-48, wherein the antibody comprises: (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) VH comprising the amino acid sequence of SEQ ID NO: 41, and VL comprising the amino acid sequence of SEQ ID NO:

46.

52. The method of any one of claims 1-49, 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.

53. The method of any one of claims 1-50, wherein the antibody comprises: (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.

54. The method of any one of claims 1-50, 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.

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

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

57. The anti-TMPRSS6 antibody of any one of claims 1-53, wherein the antibody specifically binds to human TMPRSS6.

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

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