CN118078985A

CN118078985A - Anti-TMPRSS 6 antibodies and uses thereof

Info

Publication number: CN118078985A
Application number: CN202310464524.5A
Authority: CN
Inventors: 杜欣; 陈布新; 王玉
Original assignee: Maxwell Usa Biotherapy Co ltd
Current assignee: Maxwell Usa Biotherapy Co ltd
Priority date: 2022-11-25
Filing date: 2023-04-26
Publication date: 2024-05-28

Abstract

The present application relates to anti-TMPRSS 6 antibodies and uses thereof. Antibodies and antigen binding fragments thereof that bind to type II transmembrane serine protease 6 (TMPRSS 6) on the cell surface and increase hepcidin expression are provided, as well as methods of treating iron metabolic disorders and myeloproliferative disorders using anti-TMPRSS 6 antibodies and fragments.

Description

Anti-TMPRSS 6 antibodies and uses thereof

Technical Field

The present invention relates to antibodies and antigen-binding fragments that bind TMPRSS6, and the use of antibodies and antigen-binding fragments that bind TMPRSS6 for the treatment of conditions including iron metabolic conditions and myeloproliferative neoplasms.

Background

Type II transmembrane serine protease 6 (TMPRSS 6) is encoded by the TMPRSS6 gene and is expressed predominantly in the liver. The structure of TMPRSS6 comprises a type II transmembrane domain followed by a SEA urchin sperm protein, intestinal peptidase and collectin (SEA) domain (a stem region containing two complement factors C1r/C1 s), a SEA urchin embryo growth factor and bone morphogenic protein (CUB) domain and three Low Density Lipoprotein Receptor (LDLR) class a repeat sequences and a C-terminal trypsin-like serine protease domain (Wang, c. -y et al, pharmacological front edge (front. Pharmacol.)) 2014.5:114. The alias name of TMPRSS6 (EC 3.4.21) comprises: interstitial protease-2; transmembrane protease serine 6; membrane-bound chimeric serine protease interstitial protease-2; MT2.

TMPRSS6 plays a significant role in iron homeostasis by regulating BMP-SMAD signaling pathways expressed by hepcidin, a hormone that controls iron absorption and iron storage mobilization. Hepcidin (also known as HAMP (hepcidin antimicrobial protein or peptide), encoded by HAMP in humans and non-human primates and HAMP in mice and rats, regulates systemic iron homeostasis by controlling the functional activity of the only iron efflux channel membrane iron transporter. Hepcidin can reduce plasma iron content by binding to membrane iron transporters and causing internalization and degradation of the complex, thereby preventing iron absorption at the small intestine and release of stored iron. Long-term increases in hepcidin content cause systemic iron deficiency, and hepcidin deficiency can cause systemic iron overload.

TMPRSS6 down regulates hepcidin production by a transmembrane signaling pathway triggered by iron deficiency and represses HAMP activation (Du), x. Et al Science 2008.320:1088-1092; wang (Wang), c. -y. Et al pharmacological front 2014.5:114). Low blood iron content triggers this pathway to reduce hepcidin production, which allows more iron from the diet to be absorbed through the intestine and transported from the storage site into the blood stream. In rats with acute iron deficiency, liver TMPRSS6 protein content is up-regulated, resulting in repression of hepcidin expression (Wang,) C.—Y. Et al, pharmacological front 2014.5:114. Mutations in the entire TMPRSS6 molecule (and in particular in the extracellular domain) have been identified in individuals with iron-deficiency anaemia, in particular iron-refractory iron-deficiency anaemia (IRIDA) that is non-responsive to oral iron therapy and only partially responsive to parenteral iron therapy (Wang), c. -y et al, pharmacological front 2014.5:114. Loss of function mutations in TMPRSS6 in humans lead to elevated levels of hepcidin and iron deficiency anaemia (cal Ma Sikai pull (CAMASCHELLA), c. (j. New england medical journal (NEngl Journal Med),. 2013.168:24), because overproduction of hepcidin leads to inadequate iron absorption and utilization.

Iron overload conditions result when excess iron accumulates in tissues and organs to the point where it disrupts its normal function. Iron toxicity is a common complication of iron overload conditions, leading to high mortality due to iron accumulation in major organs. Beta-thalassemia is an iron overload condition that occurs when mutations in the HBB gene cause reduced or absent production of beta-hemoglobin (beta-globin), which causes apoptosis of erythroblasts and insufficient mature erythrocytes, resulting in ineffective erythropoiesis, which causes anemia and excessive iron absorption leading to iron toxicity. In patients with beta-thalassemia, hepcidin is abnormally inhibited relative to the patient's iron load state, producing a hepcidin deficiency, which in turn allows for excessive iron absorption and development of systemic iron overload. Other conditions, such as myelodysplastic syndrome (MDS), abnormal erythropoiesis anemia, ineffective erythropoiesis in iron granulocytic anemia, are also characterized by low hepcidin, which leads to iron overload. Hemochromatosis (e.g., type 1 hemochromatosis or hereditary hemochromatosis) is an iron overload condition characterized by a pathological increase in excessive intestinal absorption of dietary iron and systemic iron storage. Current standard of care for treating iron overload conditions include transfusion for ineffective erythropoiesis, which can further exacerbate iron overload, iron chelation with poor patient compliance, and phlebotomy or splenectomy to control symptoms. Current methods of treatment under development include gene therapies targeting the HBB gene, gene therapies and gene editing targeting other related genes, hepcidin mimics, fc fusion proteins targeting TGF superfamily ligands to inhibit SMAD signaling, antisense RNA drugs targeting TMPRSS6 (e.g., ebes (El-Beshlawy) a, et al, blood Cells, molecules and diseases (Blood Cells, molecules AND DISEASES), 2019.76:53-58), and iRNA drugs targeting TMPRSS 6.

Polycythemia vera (Polycythemia vera; PV) is a chronic myeloproliferative neoplasm with constitutive activation of the JAK2/STAT5 signaling pathway, leading to increased erythrocyte mass and erythrocyte proliferation. The main cause of death may be due to thrombotic complications caused by the high viscosity of blood. Possible downstream conditions when JAK2/STAT5 signaling is constitutively activated may include parallel aberrant erythropoiesis, inflammatory environments, reduced systemic iron concentrations, and possibly altered hypoxia responses that may directly affect iron absorption in certain tissues, any or all of which may play a role in the iron metabolism of PV. (Jin Cibao (Ginzburg), Y.Z. et al, leukemia 2018.32:2105-2116). Evidence suggests that systemic iron deficiency or iron limitation targeting erythrocytes may be beneficial in reducing erythrocytosis and normalizing the hematocrit in PV.

Disclosure of Invention

The present invention relates to novel antibodies and antigen-binding fragments thereof that bind TMPRSS6, and methods of making and using antibodies and antigen-binding fragments thereof that bind TMPRSS 6.

The invention provides anti-TMPRSS 6 antibodies, nucleic acids encoding anti-TMPRSS 6 antibodies, and methods of making and using anti-TMPRSS 6 antibodies. anti-TMPRSS 6 antibodies as disclosed herein encompass anti-TMPRSS 6 antibodies and fragments thereof capable of binding TMPRSS6. anti-TMPRSS 6 antibodies as disclosed herein are capable of binding to human TMPRSS6 on the surface of cells expressing human TMPRSS6. The present invention provides anti-TMPRSS 6 antibodies for therapeutic and diagnostic use. anti-TMPRSS 6 antibodies as disclosed herein are useful for treating iron metabolic disorders, such as iron overload disorders, particularly beta-thalassemia, including (but not limited to) non-transfusion dependent thalassemia, and other disorders that do not have erythropoiesis. anti-TMPRSS 6 antibodies as disclosed herein are useful for treating myeloproliferative disorders, such as Polycythemia Vera (PV) characterized by polycythemia and erythrosis.

In one aspect, anti-TMPRSS 6 antibodies are provided that are 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 associated with the function of TMPRSS 6. In certain embodiments, an anti-TMPRSS 6 antibody disclosed herein is capable of modulating the activity of at least one component involved in modulating hepcidin expression. In certain embodiments, an anti-TMPRSS 6 antibody disclosed herein is capable of substantially inhibiting the repression of hepcidin expression by TMPRSS 6. In certain embodiments, an anti-TMPRSS 6 antibody disclosed herein is capable of increasing hepcidin expression. In certain embodiments, an anti-TMPRSS 6 antibody disclosed herein is capable of increasing the activity of a hepcidin promoter. In certain embodiments, an anti-TMPRSS 6 antibody disclosed herein is capable of substantially inhibiting TMPRSS6 repression of BMP/SMAD pathway induced hepcidin expression. anti-TMPRSS 6 antibodies disclosed herein may modulate hepcidin expression in a dose-dependent manner, including, but not limited to, substantially inhibiting TMPRSS6 repression of hepcidin expression, increasing hepcidin promoter activity, or substantially inhibiting TMPRSS6 repression of BMP/SMAD pathway-induced hepcidin expression. In certain embodiments, an anti-TMPRSS 6 antibody disclosed herein is capable of modulating hepcidin expression in a dose-dependent manner. In certain embodiments, an anti-TMPRSS 6 antibody disclosed herein is capable of increasing serum hepcidin levels in a dose-dependent manner when administered to an individual. In certain embodiments, an anti-TMPRSS 6 antibody disclosed herein is capable of reducing serum iron content in a dose-dependent manner when administered to an individual. In certain embodiments, an anti-TMPRSS 6 antibody disclosed herein is capable of increasing hepcidin RNA content in a dose-dependent manner when administered to an individual. In certain embodiments, an anti-TMPRSS 6 antibody disclosed herein is capable of reducing liver non-protoheme iron, increasing serum hepcidin, increasing liver hepcidin RNA, reducing splenomegaly, increasing red cell count (RBC), increasing Hematocrit (HCT), reducing red cell distribution width (RDW), and increasing production of mature red blood cells (increasing erythropoiesis) when administered to an individual known or suspected to have an iron overload disorder, particularly beta-thalassemia. In certain embodiments, an anti-TMPRSS 6 antibody disclosed herein is capable of reducing RBCs, reducing HCT, reducing Heme (HGB), reducing mean red blood cell volume (MCV), and reducing RDW when administered to an individual known or suspected to have a myeloproliferative disorder, such as a myeloproliferative tumor, particularly Polycythemia Vera (PV).

In another aspect, an anti-TMPRSS 6 antibody disclosed herein exhibits cross-reactivity with at least one non-human TMPRSS6. In certain embodiments, an anti-TMPRSS 6 antibody disclosed herein is 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-TMPRSS 6 antibodies disclosed herein may be capable of binding human TMPRSS6 and mouse TMPRSS6. The anti-TMPRSS 6 antibodies disclosed herein may be capable of binding to human TMPRSS6 and cynomolgus TMPRSS6. The anti-TMPRSS 6 antibodies disclosed herein may be capable of binding to each of human TMPRSS6, mouse TMPRSS6, and cynomolgus TMPRSS6.

In another aspect, the anti-TMPRSS 6 antibodies disclosed herein specifically bind to TMPRSS6 (interstitial protease-2). In certain embodiments, the anti-TMPRSS 6 antibodies disclosed herein bind to TMPRSS6 (interstitial protease-2) and do not exhibit detectable binding to the interstitial protease homolog. In certain embodiments, the anti-TMPRSS 6 antibodies disclosed herein bind to human TMPRSS6 (interstitial protease-2) and do not exhibit detectable binding to human interstitial protease-1 (ST 14). In certain embodiments, the anti-TMPRSS 6 antibodies disclosed herein bind to human TMPRSS6 (interstitial protease-2) and do not exhibit detectable binding to human interstitial protease-3 (TMPRSS 7). In certain embodiments, an anti-TMPRSS 6 antibody disclosed herein binds to human TMPRSS6 (interstitial protease-2) and does not exhibit detectable binding to either human interstitial protease-1 (ST 14) or human interstitial protease-3 (TMPRSS 7).

The anti-TMPRSS 6 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, nanobody), or other TMPRSS6 binding fragments or variants. In certain embodiments, an anti-TMPRSS 6 antibody disclosed herein may include a framework in which amino acids have been substituted into existing antibody frameworks, particularly frameworks that affect properties such as antigen binding capacity. In certain embodiments, an anti-TMPRSS 6 antibody disclosed herein may comprise Complementarity Determining Regions (CDRs) from a source (parent) antibody that have been grafted (fused) into frameworks, especially recipient human frameworks, from different types (classes) of antibodies and/or organisms other than the parent antibody. In certain embodiments, an anti-TMPRSS 6 antibody disclosed herein may include a framework in which amino acids have been substituted, mutated, or substituted in regions outside of the CDRs to affect, for example, the characteristics of antigen binding or antibody structure, e.g., in the variable region framework surrounding the CDRs and/or in the constant region (particularly the Fc region). In certain embodiments, one or more of the CDRs have been substituted, mutated, or substituted. In certain embodiments, an anti-TMPRSS 6 antibody disclosed herein may be a humanized anti-TMPRSS 6 antibody variant.

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

In one embodiment, an anti-TMPRSS 6 antibody disclosed herein comprises a Heavy Chain (HC) variable region polypeptide of the amino acid sequence set forth in SEQ ID NO. 1 or a sequence substantially identical to SEQ ID NO. 1, and a Light Chain (LC) variable region polypeptide of the amino acid sequence set forth in SEQ ID NO. 6 or a sequence substantially identical to SEQ ID NO. 6. In one embodiment, an anti-TMPRSS 6 antibody disclosed herein comprises a heavy chain complementarity determining region 1 (HC CDR 1) having amino acid sequence GYTFTSYW set forth in SEQ ID NO. 2, a heavy chain complementarity determining region 2 (HC CDR 2) having amino acid sequence IYPGSGST set forth in SEQ ID NO.3, a heavy chain complementarity determining region 3 (HC CDR 3) having amino acid sequence APYDSDYAMDY set forth in SEQ ID NO. 4; light chain complementarity determining region 1 (LC CDR 1) having amino acid sequence QDINNY set forth in SEQ ID No. 7, light chain complementarity determining region 2 (LC CDR 2) having amino acid sequence RAN set forth in SEQ ID No. 8, light chain complementarity determining region 3 (LC CDR 3) having amino acid sequence LQYDEFPLT set forth in SEQ ID No. 9; or a variant of said antibody comprising 1, 2, 3,4, 5 or 6 amino acid substitutions in the CDR regions. In one non-limiting embodiment, the anti-TMPRSS 6 antibodies disclosed herein are antibodies identified herein as MWTx-001, comprising 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.

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

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

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

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

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

In another aspect, anti-TMPRSS 6 antibodies (including variants and fragments as disclosed herein) are provided that are useful for treating iron metabolic disorders, such as iron overload disorders, particularly beta-thalassemia and other disorders of ineffective erythropoiesis. Methods and compositions are provided for using anti-TMPRSS 6 antibodies as disclosed herein for therapeutic uses including, but not limited to, the treatment of iron metabolic disorders, such as iron overload disorders, particularly beta-thalassemia and other disorders of ineffective erythropoiesis. In certain embodiments, pharmaceutical compositions comprising an anti-TMPRSS 6 antibody disclosed herein and a suitable carrier and/or excipient are provided.

In another aspect, methods for treating a disorder of iron metabolism are provided, such methods comprising administering to a subject in need thereof an effective amount of an anti-TMPRSS 6 antibody disclosed herein, wherein the administration of the effective amount of the anti-TMPRSS 6 antibody modulates the activity of a component involved in iron metabolism. In certain embodiments, a method for treating an iron overload disorder comprises administering an effective amount of an anti-TMPRSS 6 antibody disclosed herein, wherein the administration of the effective amount of the anti-TMPRSS 6 antibody modulates the activity of a component involved in iron metabolism. In certain embodiments, a method for treating an iron overload disorder comprises administering an effective amount of an anti-TMPRSS 6 antibody disclosed herein, wherein administering the effective amount of the anti-TMPRSS 6 antibody modulates the activity of at least one component involved in modulating hepcidin expression. In certain embodiments, the method comprises administering an effective amount of an anti-TMPRSS 6 antibody that inhibits repression of hepcidin expression by TMPRSS 6. In certain embodiments, administering an effective amount of an anti-TMPRSS 6 antibody increases hepcidin expression. In certain embodiments, the method comprises administering an effective amount of an anti-TMPRSS 6 antibody that increases the activity of a hepcidin promoter. In certain embodiments, the method comprises administering an effective amount of an anti-TMPRSS 6 antibody that inhibits TMPRSS6 repression of BMP/SMAD pathway induced hepcidin expression. In certain embodiments, the method comprises administering to the subject an effective amount of an anti-TMPRSS 6 antibody that results in one or more biological effects associated with an iron overload disorder, including, but not limited to, decreasing serum iron, decreasing liver non-protoheme iron, increasing serum hepcidin, increasing hepcidin RNA, decreasing splenomegaly, increasing red blood cell count (RBC), increasing Hematocrit (HCT), decreasing red cell distribution width (RDW), and/or increasing production of mature red blood cells (increasing erythropoiesis).

In another aspect, methods are provided for treating a disease or disease condition that is involved in the abnormal repression of hepcidin expression, such methods comprising administering to a subject in need thereof an effective amount of an anti-TMPRSS 6 antibody disclosed herein, wherein administration of the effective amount of the anti-TMPRSS 6 antibody modulates the activity of at least one component involved in the abnormal repression of hepcidin expression and reduces the abnormal repression of hepcidin expression. In certain embodiments, the method results in increased hepcidin expression.

In another aspect, methods for treating a disorder of iron metabolism associated with a suppressed level of hepcidin are provided, such methods comprising administering to a subject in need thereof an effective amount of an anti-TMPRSS 6 antibody disclosed herein, wherein administration of the effective amount of the anti-TMPRSS 6 antibody modulates activity of at least one component involved in suppressing the level of hepcidin. In certain embodiments, the method comprises administering an effective amount of an anti-TMPRSS 6 antibody that increases serum hepcidin levels, increases liver hepcidin RNA, and decreases serum iron levels.

In another aspect, methods are provided for treating disorders of iron metabolism, including disorders associated with and/or characterized by ineffective erythropoiesis, which may include, but are not limited to, beta-thalassemia. According to this aspect, such methods comprise administering to a subject known or suspected to have an iron metabolic disorder associated with and/or characterized by ineffective erythropoiesis an effective amount of an anti-TMPRSS 6 antibody disclosed herein, wherein the administration results in one or more changes associated with iron metabolism and/or erythropoiesis in the subject. In certain embodiments, methods are provided wherein an effective amount of an anti-TMPRSS 6 antibody is administered to treat or ameliorate at least one biological effect or symptom associated with a disorder. In particular embodiments, the methods of practice result in one or more changes, including, but not limited to, decreasing liver non-protoheme iron, increasing serum hepcidin, increasing liver hepcidin RNA, decreasing splenomegaly, increasing red blood cell count (RBC), increasing Hematocrit (HCT), decreasing red cell distribution width (RDW), and increasing mature red blood cell production (increasing erythropoiesis).

In another aspect, methods are provided for treating myeloproliferative disorders including, but not limited to, myeloproliferative neoplasms having a constitutive activation JAK2/STAT5 signaling pathway, myeloproliferative disorders characterized by increased erythrocyte mass and erythrocyte proliferation, polycythemia Vera (PV), and/or disorders characterized by polycythemia and erythrocyte proliferation. According to this aspect, such methods comprise administering to an individual known or suspected to have a myeloproliferative disorder an effective amount of an anti-TMPRSS 6 antibody disclosed herein. In certain embodiments, methods are provided wherein an effective amount of an anti-TMPRSS 6 antibody is administered to treat or ameliorate at least one biological effect or symptom associated with the disorder. In particular embodiments, practicing the methods results in one or more changes, including, but not limited to, reduction of RBCs, reduction of HCT, reduction of Heme (HGB), reduction of mean red blood cell volume (MCV), and reduction of RDW when administered to an individual known or suspected of having a myeloproliferative disorder. In a particular embodiment, practicing the method results in one or more changes, including, but not limited to, reduction of RBCs, reduction of HCT, reduction of Heme (HGB), reduction of mean red blood cell volume (MCV), and reduction of RDW when administered to an individual known or suspected to have Polycythemia Vera (PV).

In another aspect, methods for diagnosing or screening for an iron overload disorder in an individual are provided. In certain embodiments, the methods comprise administering an anti-TMPRSS 6 antibody to an individual known or suspected of having an iron overload disorder and measuring one or more biological effects or symptoms associated with the iron overload disorder.

In another aspect, methods for diagnosing or screening an individual for a myeloproliferative disorder are provided. In certain embodiments, the methods comprise administering an anti-TMPRSS 6 antibody to an individual known or suspected of having a myeloproliferative disorder and measuring one or more biological effects or symptoms associated with the myeloproliferative disorder.

In another aspect, one or more isolated nucleic acid molecules encoding at least a portion of at least one of the anti-TMPRSS 6 antibodies disclosed herein are provided. In certain embodiments, an isolated nucleic acid molecule encoding at least a portion of at least one of the anti-TMPRSS 6 antibodies disclosed herein comprises a nucleotide sequence as set forth in table 1, table 2, or table 3, or a sequence that is substantially identical (e.g., at least 85%, 90%, 92%, 95%, 97%, or 98%, 99% identical) to a nucleotide sequence as set forth in table 1, table 2, or table 3. In certain embodiments, an isolated nucleic acid molecule encoding at least one of the Heavy Chain (HC) sequences of an anti-TMPRSS 6 antibody disclosed herein may comprise a nucleotide sequence selected from at least one of: SEQ ID NO. 5 or a sequence substantially identical to SEQ ID NO. 5; 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: a sequence of SEQ ID NO. 35 substantially identical to SEQ ID NO. 35; SEQ ID NO. 45 or a sequence substantially identical to SEQ ID NO. 45; SEQ ID NO. 55 or a sequence substantially identical to SEQ ID NO. 55; SEQ ID NO. 62 or a sequence substantially identical to SEQ ID NO. 62; SEQ ID NO. 66 or a sequence substantially identical to SEQ ID NO. 66; SEQ ID NO. 70 or a sequence substantially identical to SEQ ID NO. 70; SEQ ID NO. 74 or a sequence substantially identical to SEQ ID NO. 74; SEQ ID NO. 78 or a sequence substantially identical to SEQ ID NO. 78, or SEQ ID NO. 82 or a sequence substantially identical to SEQ ID NO. 82. In certain embodiments, an isolated nucleic acid molecule encoding at least one of the Light Chain (LC) sequences of an anti-TMPRSS 6 antibody or antigen-binding fragment thereof disclosed herein may comprise a nucleotide sequence selected from at least one of: 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. 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.

In another aspect, a vector is provided comprising one or more nucleic acid molecules encoding at least one amino acid sequence of an anti-TMPRSS 6 antibody disclosed herein. In certain embodiments, vectors are provided that include one or more nucleic acid molecules encoding at least one of the Heavy Chain (HC) or Light Chain (LC) sequences of an anti-TMPRSS 6 antibody disclosed herein. In certain embodiments, vectors are provided that include a nucleic acid molecule encoding at least a portion of at least one of the amino acid sequences as set forth in table 1, table 2, or table 3, or at least a portion of an amino acid sequence that is substantially identical to an amino acid sequence as set forth in table 1, table 2, or table 3. In certain embodiments, vectors are provided that include a nucleic acid molecule encoding at least a portion of at least one of the HC or LC sequences as set forth in table 1, table 2, or table 3, or at least a portion of an amino acid sequence substantially identical to at least one of the HC or LC sequences as set forth in table 1, table 2, or table 3.

In another aspect, there is provided at least one host cell containing a vector comprising one or more nucleic acid molecules encoding the amino acid sequences of the anti-TMPRSS 6 antibodies disclosed herein. In certain embodiments, a host cell is provided that contains a vector comprising a nucleic acid molecule encoding at least a portion of at least one of the HC or LC sequences as set forth in table 1, table 2, or table 3, or at least a portion of an amino acid sequence substantially identical to at least one of the HC or LC sequences as set forth in table 1, table 2, or table 3. In certain embodiments, at least one host cell is capable of supporting vector expression and recombinant production of the anti-TMPRSS 6 antibody or antigen-binding fragment thereof encoded by the vector. In certain embodiments, at least one host cell is capable of supporting vector expression and recombinant production of an anti-TMPRSS 6 antibody or antigen-binding fragment thereof encoded by a vector comprising a nucleic acid molecule encoding at least a portion of at least one of the HC or LC sequences as set forth in table 1, table 2, or table 3 or at least a portion of an amino acid sequence substantially identical to at least one of the HC or LC sequences as set forth in table 1, table 2, or table 3. In certain embodiments, the host cell is transiently transfected with a vector comprising one or more nucleic acid molecules encoding the amino acid sequences of the anti-TMPRSS 6 antibodies or antigen-binding fragments thereof disclosed herein, wherein the host cell is capable of supporting vector expression and recombinant production of the anti-TMPRSS 6 antibodies or antigen-binding fragments thereof encoded by the vector.

Drawings

FIG. 1 shows the results of a cascade screen for anti-TMPRSS 6 antibodies, wherein antibodies that bind to human TMPRSS6 were assessed using an in vitro functional assay for HAMP promoter activity, and antibodies that have an effect on HAMP promoter activity were assessed for cross-reactivity with non-human TMPRSS 6.

Figures 2A to 2F show the effect of anti-TMPRSS 6 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 results using anti-TMPRSS 6 antibodies, and open squares represent results using the same concentration of mouse IgG or human IgG1 as negative (non-specific binding) controls. FIG. 2A shows the effect of MWTx-001 anti-TMPRSS 6 antibodies on HAMP promoter activity over a range of antibody concentrations. FIG. 2B shows the effect of MWTx-002 anti-TMPRSS 6 antibody on HAMP promoter activity over a range of antibody concentrations. FIG. 2C shows the effect of MWTx-003 anti-TMPRSS 6 antibodies on HAMP promoter activity over a range of antibody concentrations. FIG. 2D shows the effect of hzMWTx-001Var anti-TMPRSS 6 antibodies on HAMP promoter activity over a range of antibody concentrations. FIG. 2E shows the effect of hzMWTx-002Var anti-TMPRSS 6 antibodies on HAMP promoter activity over a range of antibody concentrations. FIG. 2F shows the effect of hzMWTx-003Var anti-TMPRSS 6 antibodies on HAMP promoter activity over a range of antibody concentrations.

Figures 3A to 3M show the results of the determination of the binding affinity of anti-TMPRSS 6 antibodies. Figures 3A to 3F show the results of determining the binding affinity of anti-TMPRSS 6 antibodies to human TMPRSS6 expressed on HEK293T cells using two different methods. In each graph, open circles represent results using anti-TMPRSS 6 antibodies in a range of concentrations, and open squares represent results using the same concentration of mouse IgG as a negative control. Figures 3A-3C show the results of using cell surface ELISA (measuring HRP-labeled secondary antibodies) to measure the binding of MWTx-001 (figure 3A), MWTx-002 (figure 3B) and MWTx-003 (figure 3C) to human TMPRSS6, with EC ₅₀ values calculated for each antibody used as an assessment of binding affinity. Figures 3D to 3F show the results of using FACS (measuring APC-bound secondary antibodies) to measure binding of MWTx-001 (figure 3D), MWTx-002 (figure 3E) and MWTx-003 (figure 3F) to human TMPRSS6, with EC ₅₀ values calculated for each antibody used as an assessment of binding affinity. Figures 3G to 3M show useRED96e measures the results of anti-TMPRSS 6 antibody affinity and binding kinetics against human ecto-TMPRSS6-FLAG, with analyte concentrations of 50nM, 25nM, 12.5nM, 6.25nM, 3.13nM, 1.56nM and 0.78nM. FIG. 3G shows the binding kinetics of MWTx-001 anti-TMPRSS 6 antibodies to ecto-TMPRSS 6-FLAG. FIG. 3H shows the binding kinetics of MWTx-002 anti-TMPRSS 6 antibody against ecto-TMPRSS 6-FLAG. FIG. 3I shows the binding kinetics of MWTx-003 anti-TMPRSS 6 antibody to ecto-TMPRSS 6-FLAG. FIG. 3J shows the binding kinetics of hzMWTx-001Var anti-TMPRSS 6 antibodies against ecto-TMPRSS 6-FLAG. FIG. 3K shows the binding kinetics of hzMWTx-002Var anti-TMPRSS 6 antibody against ecto-TMPRSS 6-FLAG. FIG. 3L shows the binding kinetics of hzMWTx-003Var anti-TMPRSS 6 antibody against ecto-TMPRSS 6-FLAG. Figure 3M outlines affinity measurements for all anti-TMPRSS 6 antibodies.

Fig. 4A to 4U show the measurement results of the cross-reactivity of the anti-TMPRSS 6 antibody. FIGS. 4A through 4I show the results of assays for cross-reactivity of anti-TMPRSS 6 antibodies MWTx-001, MWTx-002, and MWTx-003 against human TMPRSS6 and non-human TMPRSS6 expressed on HEK293T cells. Each histogram shows FACS results (thin line and lighter fill; indicated by antibody name) of a single antibody incubated with HEK293T cells expressing TMPRSS6 target and FACS results (thick line, darker fill) of the same antibody incubated with control HEK293T cells that do not express TMPRSS6 protein; indicated with Ctrl). FIGS. 4A-4C show the results using HEK293T cells stably expressing human TMPRSS6 (HuTMPRSS- (His) ₆) with MWTx-001 (FIG. 4A), MWTx-002 (FIG. 4B) and MWTx-003 (FIG. 4C). FIGS. 4D-4F show the results using HEK293T cells stably expressing mouse TMPRSS6 (MoTMPRSS- (His) ₆) with MWTx-001 (FIG. 4D), MWTx-002 (FIG. 4E) and MWTx-003 (FIG. 4F). FIGS. 4G through 4I show the results using HEK293T cells transiently expressing cynomolgus TMPRSS6 (CynoTMPRSS 6- (His) ₆) with MWTx-001 (FIG. 4G), MWTx-002 (FIG. 4H) and MWTx-003 (FIG. 4I). FIGS. 4J to 4U show the results of cross-reactivity of anti-TMPRSS 6 antibodies against non-human (mouse (FIGS. 4J, 4L, 4N, 4P, 4R, 4T) or cynomolgus macaque (FIGS. 4K, 4M, 4O, 4Q, 4S, 4U)) TMPRSS6 expressed on HEK293T cells, cell surface ELISA (measuring HRP-labeled secondary antibodies) was used to measure MWTx-001 anti-TMPRSS 6 antibodies (FIGS. 4J to 4K), MWTx-002 anti-TMPRSS 6 antibodies (FIGS. 4L to 4M), MWTx-003 anti-TMPRSS 6 antibodies (FIGS. 4N to 4O), a, hzMWTx-001Var anti-TMPRSS 6 antibodies (FIGS. 4P through 4Q), hzMWTx-002Var anti-TMPRSS 6 antibodies (FIGS. 4R through 4S), and hzMWTx-003Var anti-TMPRSS 6 antibodies (FIGS. 4T through 4U) in combination with non-human TMPRSS 6. In each graph, open circles represent results using anti-TMPRSS 6 antibodies, and open squares represent results of mouse IgG or human IgG1 as negative (non-specific binding) controls, with EC ₅₀ values calculated for each antibody used as an assessment of binding affinity.

FIGS. 5A-5R show the results of FACS analysis of the binding of anti-TMPRSS 6 monoclonal antibodies MWTx-001 (FIGS. 5A-5C), MWTx-002 (FIGS. 5D-5F), MWTx-003 (FIGS. 5G-5I) anti-TMPRSS 6 antibodies and humanized variants hzMWTx-001Var (FIGS. 5J-5L), hzMWTx-002Var (FIGS. 5M-5O), hzMWTx-003Var (FIGS. 5P-5R) anti-TMPRSS 6 antibodies to HEK293T cells expressing homologous mesenchymal proteases. HEK293T cells stably expressing human TMPRSS6 (interstitial protease-2) (fig. 5A, 5D, 5G, 5J, 5M, 5P) were used as positive controls and HEK293T cells overexpressing interstitial protease (ST 14) (fig. 5B, 5E, 5H, 5K, 5N, 5Q) and/or interstitial protease-3 (TMPRSS 7) (fig. 5C, 5F, 5I, 5L, 5O, 5R) proteins were used to test binding to homologous interstitial proteases. In each of the figures (fig. 5A to 5R), HEK293T cells not expressing the interstitial protease (HEK 293T) were used as negative controls, with control (Ctrl) results explicitly indicated.

Figures 6A to 6L show that anti-TMPRSS 6 antibody treatment increased hepcidin expression in mice in a dose-dependent manner. Figures 6A to 6C show the effect of MWTx-003 anti-TMPRSS 6 antibody (figures 6A to 6B) or a humanized variant hzMWTx-003Var anti-TMPRSS 6 antibody (figure 6C) thereof on serum iron. FIG. 6D shows the effect of GFP-TMPRSS6 on serum hepcidin. Figures 6D to 6F show the effect of MWTx-003 anti-TMPRSS 6 antibody (figures 6D to 6E) or a humanized variant hzMWTx-003Var anti-TMPRSS 6 antibody (figure 6F) thereof on serum iron. FIG. 6G shows the effect of GFP-TMPRSS6 on hepcidin RNA. FIGS. 6G-6I show the effect of MWTx-003 anti-TMPRSS 6 antibodies (FIGS. 6G-6H) or a humanized variant hzMWTx-003Var anti-TMPRSS 6 antibody (FIG. 6I) on hepcidin RNA. FIGS. 6J to 6L show serum concentrations of MWTx-003 anti-TMPRSS 6 antibody (FIGS. 6J to 6K) or a humanized variant hzMWTx-003Var anti-TMPRSS 6 antibody (FIG. 6L) thereof. Mouse IgG2B (MoIG B) (fig. 6A to 6B, 6D to 6E, 6G to 6H, 6J to 6K) or human IgG1 (HuIGg 1) (fig. 6C, 6F, 6I, 6L) was used as isotype control, PBS as vehicle control, and GFP vector as vector control (fig. 6A, 6D, 6G, 6J).

Figures 7A to 7R show in vivo efficacy of anti-TMPRSS 6 antibodies using a mouse model of β -thalassemia. Figures 7A-7D show the effect of using Th3/+ mice, MWTx-003 anti-TMPRSS 6 antibodies on RBCs (figure 7A), HGBs (figure 7B), HCTs (figure 7C) and RDWs (figure 7D). FIG. 7E shows the effect of using Th3/+ mice, MWTx-003 anti-TMPRSS 6 antibodies on spleen weight. FIG. 7F shows the effect of using Th3/+ mice, MWTx-003 anti-TMPRSS 6 antibodies on serum iron. FIG. 7G shows the effect of using Th3/+ mice, MWTx-003 anti-TMPRSS 6 antibodies on liver non-protoheme iron. FIG. 7H shows the effect of using Th3/+ mice, MWTx-003 anti-TMPRSS 6 antibodies on serum hepcidin. FIG. 7I shows the effect of using Th3/+ mice, MWTx-003 anti-TMPRSS 6 antibodies on hepcidin RNA. FIG. 7J shows serum concentrations of MWTx-003 anti-TMPRSS 6 antibodies using Th3/+ mice. Figures 7L to 7M show the effect of using bone marrow from Th3/+ mice, MWTx-003 anti-TMPRSS 6 antibodies on erythropoiesis. Figures 7O through 7P show the effect of using spleen cells from Th3/+ mice, MWTx-003 anti-TMPRSS 6 antibodies on erythropoiesis. The representative graphs in fig. 7K-7P show four different cell clusters (I: basophils; II: polychromatic erythroblasts; III: orthochromatic erythroblasts and coreless reticulocytes and IV: mature erythrocytes) and highlight their corresponding cell number percentages. Wild-type mice were used as positive controls (fig. 7A to 7J, 7K, 7N) and mouse IgG2b (MoIgG b) was used as isotype control in therapy (fig. 7A to 7J, 7L, 7O). The bar graphs in fig. 7Q-7R show the average results of cell clusters I, II, III and IV in bone marrow (fig. 7Q) and spleen (fig. 7R) after 4 weeks for each treatment regimen (WT, th3/+w/MoIgG b, th 3/+w/MWTx-003), where the comparison allows identification of the transition of each population, most notably, to mature red blood cells (cluster IV) after MWTx-003 treatment.

Figures 8A to 8D show useRED96e, MWTx-001, MWTx-002 and MWTx-003 anti-TMPRSS 6 antibodies against epitope grouping (binding) of human ecto-TMPRSS 6-FLAG. FIG. 8A shows epitope grouping of MWTx-001 anti-TMPRSS 6 antibodies against ecto-TMPRSS 6-FLAG. FIG. 8B shows epitope grouping of MWTx-002 anti-TMPRSS 6 antibodies against ecto-TMPRSS 6-FLAG. FIG. 8C shows epitope grouping of MWTx-003 anti-TMPRSS 6 antibodies against ecto-TMPRSS 6-FLAG. FIG. 8D outlines the association signals of MWTx-001, MWTx-002, and MWTx-003 anti-TMPRSS 6 antibodies.

Figures 9A to 9H show the results of sub-chronic treatment with anti-TMPRSS 6 antibodies in a Jak2V617/+ Vav-iCre mouse model of PV, mice received IP injections at dose levels of 2mg/kg, 5mg/kg or 10mg/kg of recombinant MWTx-003 (r 4K 12B), or mouse IgG2B isotype control (MoIgG 2B) received 10mg/kg every 4 days for 3 weeks and killed 4 days after the last injection for analysis; WT mice did not receive treatment; each symbol in the drawing represents a single mouse. Fig. 9A-9C show endpoint measurements of hematology parameters HCT (fig. 9A), RBC (fig. 9B), and HGB (fig. 9C) for each treatment and dose level. Fig. 9D to 9E also show end-point measurements for each treatment and dose level, where fig. 9D shows splenomegaly (splenomegaly index measured in mg/g body weight) indicating a dose-dependent evolution of iron-limited erythropoiesis in mice treated with MWTx-003, fig. 9E shows serum hepcidin content (ng/ml), and fig. 9F shows serum anti-TMPRSS 6 concentration (μg/ml) measured by cell surface ELISA at the end of the study. Fig. 9G shows FACS results of measuring early erythrocyte precursors (I cluster basophils and II cluster polychromic erythroblasts) in bone marrow (upper row) and spleen (lower row), showing the results of WT (upper and lower row left panels), moIgG b isotype control (upper and lower row middle panels) and 10mg/kg anti-TMPRSS 6 MWTx-003 treatment (upper and lower row right panels). Fig. 9H shows Prussian blue staining (Prussian blue staining) images of liver sections (left panel) and spleen sections (right panel) from mouse IgG2b isotype control MoIgG b treatment (upper row) and increased doses of anti-TMPRSS 6 MWTx-003, indicating increased iron deposition in the spleen but no significant change in liver iron content between the treated mice and the control. Significant iron deposition is indicated by the arrow. In fig. 9A-9F, P <0.0001, P <0.001, P <0.05, were modulated using single factor ANOVA and base multiple comparison (Tukey multiple comparison adjustment).

Detailed Description

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

Terminology/definition

Unless defined otherwise, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by one of ordinary skill in the art. The use of the singular term "a," "an," or "the" or other use of the singular term includes plural referents unless the context clearly dictates otherwise, and the plural term shall include the singular. Thus, for example, reference to "an antibody" includes "one or more" antibodies "or" a plurality "of such antibodies. All publications mentioned herein are incorporated by reference in their entirety.

In general, molecular biology, microbiology, cell and tissue culture, protein and nucleotide chemistry methods, and nomenclature and techniques of recombinant DNA technology that are available to those of skill in the art can be used for the antibodies, antigen binding fragments, compositions, and methods disclosed herein. The techniques and procedures described herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references, especially Sambrook et al (1989), laboratory guidelines (MOLECULAR CLONING: A LABORATORY MANUAL) (2 nd edition, cold spring harbor laboratory publication (Cold Spring Harbor Laboratory Press), new york cold spring harbor (Cold Spring Harbor, n.y.)) and An Subei mol (Ausubel) et al (1994), molecular biology experimental guidelines (CURRENT PROTOCOLS IN MOLECULAR BIOLOGY), volumes I-III (John Wiley & Sons, n.y.)) in John Wiley international publications, new york. Unless otherwise indicated herein, enzymatic reactions and purification techniques are performed according to the manufacturer's instructions or as commonly accomplished in the art or as described herein. Techniques and methods for pharmaceutical preparations and formulations, as well as for treating individuals, are described herein using conventional nomenclature.

An "antibody" refers most broadly to a polypeptide or combination of polypeptides that recognizes and binds to an antigen through one or more immunoglobulin variable regions, where the immunoglobulin variable regions may be naturally occurring or non-naturally occurring, e.g., due to engineering, chimeric, humanization, optimization, CDR grafting, or affinity maturation.

An "antibody" as disclosed herein may be a whole (intact, full length) antibody, a single chain antibody, or an antigen binding fragment having one or both chains, and may be naturally occurring and non-naturally occurring. Antibodies include at least sufficient Complementarity Determining Regions (CDRs) interspersed with Framework Regions (FRs) for the antibody to recognize and bind to an antigen. The anti-TMPRSS 6 antibodies disclosed herein may be (but are not limited to) at least one of the following: monoclonal antibodies, recombinant monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, single chain antibodies, fab fragments, single chain variable fragments (scFv), aptamers, single domain antibodies (VHH or nanobody), recombinant antibodies, modified antibodies with peptides/other moieties linked to the antibody and/or additional amino acids added to the N-or C-terminus, or other TMPRSS6 binding fragments or variants. Whole antibodies, full length antibodies, whole antibodies, naturally occurring antibodies or equivalent terms are understood to mean polypeptides, in particular glycoproteins, comprising at least two Heavy Chains (HC) and two Light Chains (LC) interconnected by disulfide bonds. Each HC includes a heavy chain variable region (VH) and an HC constant region (CH), and each light chain includes a light chain variable region (VL) and an LC constant region (CL). The HC and LC variable regions (i.e., VH and VL) comprise binding domains that interact with antigens. VH and VL regions can be further subdivided into CDR regions characterized by hypervariability interspersed with FR regions that are typically more conserved. Each VH and VL is typically composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant regions of antibodies may mediate the binding of immunoglobulins to host tissues or factors, including the various cells of the immune system and the classical complement system. Typically, an antibody includes at least Heavy Chain (HC) CDR1, CDR2, and CDR3, and Light Chain (LC) CDR1, CDR2, and CDR3 sequences, wherein any of these sequences may be naturally or non-naturally occurring. Antibodies may include fewer CDR sequences, so long as the antibody can recognize and bind to an antigen.

The anti-TMPRSS 6 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 a nucleic acid molecule encoding such framework sequence. Variants can be constructed from HC and LC moieties that are independently derived from different sources. Techniques for producing variants include, but are not limited to, conservative amino acid substitutions, computer modeling, screening candidate polypeptides alone or in combination, and codon optimization, and it is understood that the skilled artisan is able to produce antibody variants as desired. The anti-TMPRSS 6 antibodies disclosed herein may be fragments. The antigen binding function of an antibody can be performed by, for example, fragments of: fab fragments; a monovalent fragment consisting of VL, VH, CL and CH1 domains; a F (ab) ₂ fragment; a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; fd fragment consisting of VH and CH1 domains; a single chain variable fragment (scFv) consisting of VL and VH domains of a single arm of an antibody; a single domain antibody (dAb) fragment consisting of a VH domain; and isolated CDRs (VHH, nanobody), or aptamers. Antigen binding moieties can be incorporated into single domain antibodies, maxiantibodies, miniantibodies, nanobodies, intracellular antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, v-NAR, and bi-scFv (see, e.g., hollager and Handerson (Hudson), 2005, nature Biotechnology (Nature Biotechnology), 23,9,1126-1136). The antigen-binding portion of the antibody can be grafted into a polypeptide-based scaffold to form a monofunctional antibody (see, e.g., U.S. patent No. 6,703,199, which describes fibronectin polypeptide monofunctional antibodies).

The term antibody encompasses a wide variety of classes of polypeptides that can be biochemically distinguished. "class" of antibodies refers to the type of constant domain or constant region that the heavy chain has. Those of skill in the art understand that there are five major classes of antibodies, igA, igD, igE, igG and IgM, several of which can be further divided into subclasses (isotypes), such as IgG1, igG2, igG3, igG4, igA1, and IgA2, each of which is well characterized and known to confer functional specificity. The modified forms of each of these classes and isotypes are readily discernable and within the scope of the invention. Although all immunoglobulin classes are within the scope of the invention, the invention will be directed primarily to immunoglobulin molecules of the IgG class.

The term "chimeric" antibody refers to an antibody in which a portion of the Heavy (HC) and/or Light (LC) chains involved in the formation of an immunoreactive site are derived from a particular source or species, while the remainder of the HC and/or LC are derived from a different source or species. In certain embodiments, the target binding region or site is from a non-human source (e.g., a mouse or a non-human primate), and the constant region is a human.

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), wherein CDRs from the parent, non-human antibody are grafted (fused) into a framework comprising variable regions derived from a human immunoglobulin framework, particularly a recipient human framework or a human co-framework. Techniques and principles for designing, preparing and testing humanized antibodies are known (Jones PT, dil (Dear) PH, foote J, neuberger MS, winter g. Substitution of complementarity determining regions in human antibodies with complementarity determining regions from mice (REPLACING THE-DETERMINING REGIONS IN A HUMAN ANTIBODY WITH THOSE FROM A MOUSE) & Nature (Nature) & 5, 29, 4, 1986; 321 (6069): 522-5; almag (Almagro) JC, frassen (frankson) J. Humanization of antibodies (Humanization of antibodies) & bioscience Front (Front Biosci) & 1, 2008; 13:1619-33). It will be appreciated that the receptor architecture at a variety of locations can be altered to produce humanized antibodies with improved characteristics (e.g., high affinity for the target, low clearance, low toxicity, etc.) depending on the desired use. The anti-TMPRSS 6 antibodies disclosed herein may be humanized variants.

"Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to an inherent binding affinity that 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 about 50% of maximum binding (calculated EC ₅₀) can be used as an assessment of affinity. The affinity of a molecule X for its partner Y can be generally expressed by a dissociation constant (Kd or Kd, representing k _off/k_on measured for interaction).

An "individual" is a mammal, wherein the mammal includes, but is not limited to, primates (e.g., humans and non-human primates, such as monkeys), domesticated animals (e.g., cows, sheep, cats, dogs, pigs, luo Maji horses), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the subject is a human. The phrase "to an individual in need thereof" or "to a patient in need of treatment" or "an individual in need of treatment" may comprise an individual who would benefit from administration of an anti-TMPRSS 6 antibody disclosed herein to treat an iron overload disorder. It is understood that administration of an anti-TMPRSS 6 antibody encompasses administration to an "individual in need thereof" (which may be interpreted to mean an individual known or suspected to have an iron overload disorder, particularly beta-thalassemia) based on an index such as symptoms, family history or genotype. It is further understood that anti-TMPRSS 6 antibodies may be administered to individuals not known or suspected to have an iron metabolic disorder for purposes that may include, but are not limited to, prophylactic or preventative purposes, screening, diagnostic, research purposes, or achieving a result different from that of treating the disorder.

For example, an "effective amount" of an anti-TMPRSS 6 antibody in a pharmaceutical formulation refers to an amount that is at the necessary dose and for the necessary period of time effective to achieve the desired therapeutic or prophylactic result. It will be understood that an "effective amount" is intended to refer to an amount of an anti-TMPRSS 6 antibody or a pharmaceutical composition comprising an anti-TMPRSS 6 antibody that will elicit a biological response or a desired therapeutic effect in a cell, tissue, system, non-human animal subject, non-human mammalian subject, or measured human subject. The terms "therapeutically effective amount," "pharmacologically effective amount," and "physiologically effective amount" are used interchangeably to refer to the amount of anti-TMPRSS 6 antibody required to provide a threshold level of active agent in the blood stream or in the tissue of interest. The exact amount will depend on a number of factors, such as the particular anti-TMPRSS 6 antibody (active agent); the components and physical properties of the composition; an expected population of individuals/patients to be treated; such as disease conditions, age, sex, and individual weight considerations, and the like, and may be readily determined by one of ordinary skill in the art based on 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, such as a measurement of the same individual prior to initiation of the treatment described herein, or a measurement of a control individual (or multiple control individuals) in the absence of the treatment described herein.

The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a formulation, in particular an anti-TMPRSS 6 antibody, in such a form as to allow the biological activity of the active ingredient contained therein to be effective. It will be appreciated that the pharmaceutical composition may contain more than one active ingredient, for example more than one anti-TMPRSS 6 antibody, or a combination of an anti-TMPRSS 6 antibody with another active ingredient acting on a different target, wherein such a combination may be, but is not limited to, a combination of an anti-TMPRSS 6 antibody with another active ingredient having a desired effect on the hematopoietic process, in particular erythropoiesis, a combination of an anti-TMPRSS 6 antibody with a gene therapeutic agent, for example an agent that performs gene therapy targeting the HBB gene, or a combination of an anti-TMPRSS 6 antibody with an Fc fusion protein that targets a TGF superfamily ligand to stimulate erythropoiesis. By "pharmaceutically acceptable carrier" is meant an ingredient in a pharmaceutical formulation that is not toxic to the individual other than the active ingredient. It is understood that the pharmaceutically acceptable carrier may be, but is not limited to, a buffer, excipient, stabilizer, adjuvant or preservative.

As used herein, the term "treatment" or similar terms may refer to a result that is considered beneficial to a particular individual in a defined set of circumstances. Treating an iron metabolism disorder may refer not only to any of reducing, alleviating, slowing, interrupting, arresting, alleviating, stopping, or reversing the progression or severity of an existing symptom, disorder, condition, or disease, but may further encompass preventing or delaying the onset of symptoms of one or more iron overload disorders, and/or alleviating the severity or frequency of symptoms of one or more iron overload disorders. The term "treatment" or "method of treatment" or equivalent may encompass one or more uses of the anti-TMPRSS 6 antibodies disclosed herein, including but not limited to therapeutic, prophylactic, diagnostic, imaging, and screening uses.

As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating a nucleic acid to which a vector sequence is linked in a host cell into which the vector is introduced. Vectors capable of directing the expression of their operably linked nucleic acids are referred to herein as "expression vectors".

Anti-TMPRSS 6 antibodies

Antibodies and antigen binding fragments are provided that are capable of binding to TMPRSS6 on the cell surface and modulating the activity of at least one component involved in iron metabolism, particularly at least one component involved in an iron overload disorder associated with abnormal repression of hepcidin expression. anti-TMPRSS 6 antibodies capable of binding TMPRSS6 on the cell surface and modulating the activity of at least one component involved in modulating hepcidin expression are useful in methods of treating iron overload disorders associated with abnormal repression of hepcidin expression. anti-TMPRSS 6 antibodies capable of binding TMPRSS6 on the cell surface and modulating repression of hepcidin expression by TMPRSS6 are useful for therapeutically targeting TMPRSS6 in methods of treating iron overload disorders associated with aberrant repression of hepcidin expression and/or other iron metabolic disorders.

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

It should be understood that as used herein, "modulating" or similar terms may refer to one or more effects that may occur when an anti-TMPRSS 6 antibody disclosed herein binds to its target. Depending on the component under consideration, "modulation" and its equivalents may refer to different modes of action and effects, i.e., modulation may 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 modulation may refer to increasing, enhancing, or having an agonist effect on those components.

It is understood that the term "component" may refer not only to the target molecule TMPRSS6, but also to downstream processes or pathways involved in iron metabolism. Thus, components within the meaning of a process or pathway may be, but are not limited to, one or more hematopoietic processes that regulate hepcidin expression, repression of hepcidin expression by TMPRSS6, processes of hepcidin expression, regulation of hepcidin content, increase of hepcidin content, repression of hepcidin promoter activity or BMP/SMAD pathway induced hepcidin expression by TMPRSS6, regulation of liver non-protoheme iron content, involvement in one or more processes of splenomegaly, or involvement in regulation of red blood cell count (RBC), hematocrit (HCT), red cell distribution width (RDW), and erythropoiesis (especially production of mature red blood cells).

Anti-TMPRSS 6 antibodies as disclosed herein may 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-TMPRSS 6 antibodies as disclosed herein may be used to therapeutically target at least one component involved in modulating hepcidin expression, and to modulate the activity of the component to achieve increased hepcidin expression. In certain embodiments, anti-TMPRSS 6 antibodies as disclosed herein may be used to modulate the activity of a hepcidin promoter to achieve increased hepcidin expression. It is to be understood that anti-TMPRSS 6 antibodies as disclosed herein may be used to therapeutically target TMPRSS6 and thereby regulate downstream activity of other components of hepcidin expression, including, but not limited to, modulation of liver non-protoheme iron content, involvement in one or more processes of splenomegaly, or involvement in one or more hematopoietic processes of regulation of red blood cell count (RBC), hematocrit (HCT), red blood cell distribution width (RDW), and erythropoiesis, particularly production of mature red blood cells.

The use of an anti-TMPRSS 6 antibody as disclosed herein to therapeutically target at least one component involved in iron metabolism allows for precise modulation of the targeting component. It will be appreciated that by using anti-TMPRSS 6 antibodies as disclosed herein to precisely target TMPRSS6 and its downstream effects on at least one component involved in modulating hepcidin expression, it is possible to avoid undesirable effects, delivery difficulties and/or effectiveness, and regulatory disorders associated with other methods of treating iron overload conditions currently in use or under development, such as transfusion that can further exacerbate iron overload, poor patient compliance of iron chelation, invasive phlebotomy or splenectomy that only controls symptoms, gene therapy targeting HBB genes with potential permanent pleiotropic effects in multiple systems, gene therapy and gene editing with unknown off-target effects, targeting TGF superfamily ligands to inhibit Fc fusion proteins that do not reduce SMAD signaling required for iron chelation therapies that control iron overload, and other methods of difficult control or delivery of antisense or iRNA drugs such as hepcidin mimics and targeted TMPRSS 6. It will be appreciated that the use of anti-TMPRSS 6 antibodies for precise therapeutic targeting does not exclude the possibility of using anti-TMPRSS 6 antibodies in methods and compositions for combination therapy, e.g. in combination with another active ingredient acting on a different target, in combination with antibodies binding to a different target, in combination with gene therapeutics and methods targeting the HBB gene, or in combination with Fc fusion proteins targeting TGF superfamily ligands to stimulate erythropoiesis.

The anti-TMPRSS 6 antibodies disclosed herein allow for the development of therapies (e.g., dosages, dosing frequencies) that can be tailored to each individual, where they can be easily continued and discontinued, and combined with other therapies. In certain strategic embodiments, the anti-TMPRSS 6 antibodies disclosed herein may be combined with other therapies that may address multiple therapeutic objectives and/or address the deficiencies or adverse effects of one of the therapies in a combination therapy.

Exemplary embodiments of anti-TMPRSS 6 antibodies and uses thereof

Non-limiting exemplary embodiments of anti-TMPRSS 6 antibodies of the present invention are presently disclosed, particularly in the examples, tables and figures.

A. Antibodies capable of binding TMPRSS6

As demonstrated in the examples, a functional cascade can be used to identify and characterize the anti-TMPRSS 6 antibodies of the invention, wherein a first step in the cascade comprises screening for antibodies capable of binding to human TMPRSS6 on the surface of a cell expressing TMPRSS6 (example 1, fig. 1), followed by a second step to identify antibodies capable of binding to human TMPRSS6 on the surface of a cell expressing TMPRSS6 and modulating the activity of a component involved in iron metabolism, in which case the ability to increase the activity of the Hepcidin (HAMP) promoter is tested (example 2). As demonstrated by the exemplary embodiment shown in fig. 1, the first step identified 143 antibodies (clones) capable of binding to human TMPRSS6 on the surface of TMPRSS6 expressing cells, and the second step identified ten (10) antibodies (screened out of 143) as "active" antibodies (clones) capable of increasing Hepcidin (HAMP) promoter activity.

In the third step of the functional cascade (fig. 1), ten (10) "active" antibodies were tested for cross-reactivity with non-human TMPRSS6 targets from sources that would be relevant for further studies, i.e., testing for cross-reactivity with mouse TMPRSS6 relevant for preclinical efficacy studies in a mouse model, and testing for cross-reactivity with cynomolgus macaque TMPRSS6 relevant for toxicity (safety) tests. As demonstrated by the exemplary examples shown in fig. 1, demonstrated in example 4, and illustrated in fig. 4, three (3) clones (screened out of 10) exhibited cross-reactivity with at least one non-human TMPRSS6 and were named MWTx-001, MWTx-002, and MWTx-003. Each of the monoclonal antibodies was sequenced and CDRs on each HC and LC were identified (Kabat numbering). The HC and LC sequences were 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 appreciated that depending on the isolation and sequencing of the monoclonal antibodies from the hybridoma cell line producing the monoclonal antibodies, the antibodies may be monoclonal antibodies isolated from the antibody producing cell line or recombinant monoclonal antibodies produced by recombinant expression of the antibodies of known HC and LC. The MWTx-001 monoclonal antibody-producing hybridoma cell line has been deposited at the American type culture Collection (AMERICAN TYPE Culture Collection) of university of Virginia, calif. 10801 (University Boulevard, manassas, virginia,20110,United States of America) at ATCC deposit number PTA-126759 according to Budapest treaty (the Budapest Treaty) at United states culture Collection (20110)). The MWTx-002 monoclonal antibody-producing hybridoma cell line has been deposited at the American type culture Collection/>, university of Va.mangasas, va.20110, of the United states, fast, under ATCC deposit number PTA-126760, at 5.27 of 2020The MWTx-003 monoclonal antibody-producing hybridoma cell line has been deposited at the American type culture Collection/>, university of Va.mangasas, va.20110, of the United states, fast, under ATCC deposit number PTA-126761, at 5.27 of 2020

A. Humanized variants

Humanized antibodies comprising CDRs derived from a non-human source grafted into a human derived antibody framework are expected to be non-immunogenic when administered to a human subject. As demonstrated by the exemplary embodiment disclosed in example 2, humanized anti-TMPRSS 6 antibody variants were successfully generated, tested, optimized and selected. A number of candidate HC and LC variants were developed, where each HC or LC variant has the same CDR sequence, but the variable region framework sequences can vary at more than 90% of the framework positions, and these variants were tested in different HC/LC combinations to identify combinations with the desired characteristics. After initial design and testing, variants exhibiting the desired antigen binding affinity are selected for further evaluation and development, including, but not limited to, modification of some parental CDR sequences to avoid potentially undesirable events, such as aspartic acid isomerization, and modification of some constant regions (fcs) to achieve the desired function, such as minimizing antibody-dependent cellular cytotoxicity (ADCC), to achieve humanized variants hzMWTx-001Var (SEQ ID NOs: 73 (HC) and 75 (LC)), hzMWTx-002Var (SEQ ID NOs: 77 (HC) and 79 (LC)), and hzMWTx-003Var (SEQ ID NOs: 81 (HC) and 83 (LC)).

A. anti-TMPRSS 6 antibodies that increase hepcidin promoter activity

As disclosed herein, antibodies for treating iron overload conditions characterized by reduced expression of hepcidin can modulate the activity of at least one component involved in hepcidin expression, wherein the component can be the activity of a hepcidin promoter. As demonstrated by using the exemplary examples of in vitro assays disclosed in example 2, anti-TMPRSS 6 antibodies MWTx-001, MWTx-002, MWTx-003, hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var increased HAMP promoter activity in a dose-dependent manner (fig. 2A to 2F), while isotype controls at the same concentrations did not increase HAMP promoter activity.

A. anti-TMPRSS 6 antibodies with high affinity for targets in a related biological context

Anti-TMPRSS 6 antibodies exhibit high affinity for biologically appropriate targets (i.e., human TMPRSS6 expressed on the cell surface). As demonstrated by the exemplary examples of measuring affinity using the three different methods disclosed in example 3 and fig. 3M, monoclonal antibodies MWTx-001, MWTx-002, and MWTx-003, as well as humanized variants hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var, consistently exhibited favorable affinity properties for a therapeutically effective antibody or antibody fragment.

A. anti-TMPRSS 6 antibodies cross-reactive with non-human targets

It is desirable that the therapeutically useful antibodies or antibody fragments have sufficient cross-reactivity with non-human targets (non-human homologs) from sources that will be relevant for further studies (e.g., 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, e.g., from cynomolgus macaques. As demonstrated by the exemplary embodiment disclosed in example 4, MWTx-001, hzMWTx-001Var, MWTx-003, and hzMWTx-003Var show detectable cross-reactivity with mouse TMPRSS6, while MWTx-001, MWTx-002, MWTx-003, hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var show detectable cross-reactivity with cynomolgus macaque TMPRSS 6.

A. anti-TMPRSS 6 antibodies specifically bind TMPRSS6 (interstitial protease-2)

Antibodies that have a high degree of specific binding to the target protein and low cross-reactivity with the cognate protein in the same organism are expected to have reduced off-target effects or no off-target effects. The anti-TMPRSS 6 antibodies provided herein exhibit high specificity for human TMPRSS6 (interstitial protease-2), making them suitable for use in targeting compositions and methods. As demonstrated by the exemplary embodiments disclosed in example 5 and illustrated in fig. 5A-5R, monoclonal antibodies MWTx-001, MWTx-002, and MWTx-003, and humanized variants hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var, exhibited specific binding to human TMPRSS6 (interstitial protease-2) and no detectable cross-reactivity with homologous human interstitial proteases, i.e., these antibodies did not exhibit detectable binding to either interstitial protease-1 (ST 14) or interstitial protease-3 (TMPRSS 7).

A. anti-TMPRSS 6 antibodies having in vivo dose-dependent effects on hormones and symptoms associated with iron overload disorders

Antibodies that increase the level of serum hepcidin (a hormone that controls iron absorption and mobilization from iron stores) are expected to reduce, ameliorate or prevent symptoms of iron overload disorders, particularly those that reduce, ameliorate or prevent elevated serum iron levels. As demonstrated by the exemplary embodiment shown in example 6, administration of anti-TMPRSS 6 monoclonal antibody MWTx-003 or humanized variant hzMWTx-003Var to wild-type individuals (i.e., individuals not yet known or suspected of having iron overload) resulted in increased serum hepcidin levels (fig. 6A-6C), decreased serum iron levels (fig. 6D-6F), and increased hepcidin RNA levels (fig. 6G-6I) compared to isotype controls. These effects are dose-dependent, which can be interpreted as indicating that a dose-dependent in vivo effect of an anti-TMPRSS 6 antibody without wishing to be bound by a mechanism of action indicates that the skilled person can determine an effective amount (dose) for a given individual.

A. anti-TMPRSS 6 antibodies with in vivo efficacy in a beta-thalassemia model

Antibodies and antibody fragments that can reduce symptoms of one or more iron overload disorders in vivo when administered to an individual exhibiting an animal model of a disease (i.e., an individual known or suspected of having an iron overload disorder) are expected to be therapeutically effective for clinical use. As demonstrated by the exemplary examples shown in example 7 using the Th3/+ mouse model of β -thalassemia, administration of anti-TMPRSS 6 monoclonal antibody MWTx-003 resulted in a variety of effects compared to isotype control, including, but not limited to, reduction of liver non-protoheme iron, increase of serum hepcidin, increase of liver hepcidin RNA, reduction of splenomegaly, increase of red blood cell count (RBC), increase of Hematocrit (HCT), reduction of red cell distribution width (RDW), and increase of mature red blood cell production (increased erythropoiesis). Each of these effects may be understood as ameliorating symptoms of the disorder. Symptoms of the disorder appear in a number of biological systems, including, but not limited to, effects in the liver (effects on liver non-protoheme iron, liver hepcidin RNA), effects in the blood (effects on serum iron content, circulating hormone content, especially serum hepcidin content, RBC, HCT, RDW), effects on spleen size and function (splenomegaly), and erythropoiesis in multiple sites including, but not limited to, bone marrow and spleen (effects on abundance of different precursor cell types and abundance of mature erythrocytes in erythropoiesis sites). Administration of anti-TMPRSS 6 antibodies will ameliorate a variety of symptoms in the whole disease model individual, thereby shifting the measured symptom levels from those seen in isotype controls of the disease model (untreated disease) and toward those seen in wild-type littermates, which represent normal levels in genetically similar individuals that have not yet been known or suspected of having disease. Without wishing to be bound by theory or mechanism of action, it is understood that the treatment of ineffective erythropoiesis as a driving force for aberrant hepcidin repression, resulting in increased iron absorption and iron overload, such that improved erythropoiesis and maturation into erythrocytes should be therapeutically beneficial for the treatment of iron overload conditions. Non-limiting exemplary embodiments of the invention disclose an anti-TMPRSS 6 antibody therapy that increases erythroblasts differentiation and maturation into erythrocytes and also reduces iron loading.

Anti-TMPRSS 6 antibodies with in vivo efficacy in Polycythemia Vera (PV) model

Antibodies and antibody fragments that can reduce one or more symptoms of a myeloproliferative disorder in vivo when administered to an individual exhibiting an animal model of the disease (i.e., an individual known or suspected of having a myeloproliferative disorder) are expected to be therapeutically effective for clinical use. The exemplary embodiment shown in example 9 using the Jak2V617/+ Vav-iCre mouse model of PV demonstrates that administration of anti-TMPRSS 6 recombinant monoclonal antibody MWTx-003 produces a variety of in vivo effects compared to isotype control, including, but not limited to, dose-dependent reduction of blood volume ratio (HCT) levels, reduction of circulating Red Blood Cell (RBC) counts, and a decrease in polycythemia Heme (HGB) concentration, as well as increases in hepcidin levels, decreases in serum iron levels, and differential effects on spleen and liver, wherein administration of anti-TMPRSS 6 recombinant monoclonal antibody MWTx-003 does not cause a significant change in liver iron levels, but causes a significant increase in iron deposits in spleen macrophages. Some effects are understood to be an improvement in the symptoms of the disorder. Symptoms of the disorder are manifested in a variety of biological systems, including but not limited to effects in the liver, spleen, blood (especially serum hepcidin levels, RBCs, HCTs, erythrocytosis) and bone marrow. Administration of anti-TMPRSS 6 antibodies ameliorates a variety of symptoms in individuals in the whole disease model, moving the measured symptom levels away from those seen in disease model (untreated disease) isotype controls and toward those seen in wild-type littermates, which represent normal levels in individuals that are not known or suspected of having the disease. Non-limiting exemplary embodiments of the invention disclose an anti-TMPRSS 6 antibody therapy that increases hepcidin levels and reduces erythrocytosis in individuals with PV.

Composition and method for producing the same

Compositions are provided that include a safe and effective amount of an anti-TMPRSS 6 antibody of the 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, buffers, dextrose, water, glycerol, ethanol, excipients, stabilizers, preservatives or combinations thereof. It should be understood that the pharmaceutical formulation should match the mode of administration.

The anti-TMPRSS 6 antibodies disclosed herein may be administered by any suitable means, including but not limited to injection or parenteral infusion. Parenteral infusion may include intramuscular, intravenous, intraarterial, intraperitoneal, subcutaneous administration, or parenteral delivery to the liver. The anti-TMPRSS 6 antibodies disclosed herein may be formulated for introduction into liver tissue or vascular structures for local delivery to target tissue. The anti-TMPRSS 6 antibodies disclosed herein may be administered using a device, either in a depot form, or in a sustained release formulation (e.g., semipermeable matrix or microcapsules of solid hydrophobic polymer containing the antibody), allowing slow and/or measured and/or local delivery. The anti-TMPRSS 6 antibodies disclosed herein can be formulated and administered using colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in the form of macroemulsions.

Method of

Methods of treating iron metabolism disorders using an effective amount of an anti-TMPRSS 6 antibody disclosed herein are provided. Without wishing to be bound by a particular mechanism of action, the methods provided for targeting TMPRSS6 using anti-TMPRSS 6 antibodies disclosed herein may produce a number of 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, methods are provided for treating an iron metabolic disorder using an effective amount of an anti-TMPRSS 6 antibody disclosed herein to modulate the activity of a component involved in iron metabolism. In particular, methods are provided for treating iron overload disorders associated with excessive iron accumulation in tissues and organs, including disorders associated with or characterized by ineffective erythropoiesis, which may include, but are not limited to, beta-thalassemia, particularly non-transfusion dependent thalassemia, myelodysplastic syndrome (MDS), erythropoiesis-poor anemia, and iron granulomatous anemia. Methods are provided for treating iron overload disorders associated with low hepcidin levels, particularly disorders associated with repressed hepcidin expression, including diseases or conditions involving aberrant repression of hepcidin expression, by administering an anti-TMPRSS 6 antibody capable of increasing hepcidin expression, without limitation to a single mechanism of action. Methods for treating myeloproliferative disorders are provided. Methods for treating Polycythemia Vera (PV) are provided. Methods for treating Polycythemia Vera (PV) associated with insufficient hepcidin repression are provided.

A method for treating a disorder of iron metabolism as provided herein comprises administering to a subject in need thereof an effective amount of an anti-TMPRSS 6 antibody disclosed herein, wherein administration of the effective amount of the anti-TMPRSS 6 antibody ameliorates at least one biological effect (symptom) associated with the disorder. Methods for treating an iron metabolism disorder associated with hindered statin content are provided, wherein administering an effective amount of an anti-TMPRSS 6 antibody disclosed herein to a subject in need thereof results in at least one of: increased hepcidin promoter activity, increased hepcidin transcription, increased hepcidin RNA content and increased hepcidin content (especially serum hepcidin content). Methods are provided for treating a subject known or suspected to have an iron overload disorder, wherein administration of an effective amount of an anti-TMPRSS 6 antibody results in one or more biological effects including, but not limited to, reduction of liver non-protoheme iron, increase of serum hepcidin, increase of liver hepcidin RNA, reduction of splenomegaly, increase of red blood cell count (RBC), increase of Hematocrit (HCT), reduction of red cell distribution width (RDW), and increase of mature red blood cell production (increase of erythropoiesis). Methods are provided for treating a subject known or suspected to have an iron overload disorder characterized by ineffective erythropoiesis, wherein administration of an effective amount of an anti-TMPRSS 6 antibody produces one or more biological effects, including, but not limited to, decreasing liver non-protoheme iron, increasing serum hepcidin, increasing liver hepcidin RNA, decreasing splenomegaly, increasing red blood cell count (RBC), increasing Hematocrit (HCT), decreasing red cell distribution width (RDW), and increasing production of mature red blood cells (increasing erythropoiesis).

Methods and compositions are provided for treating iron metabolic disorders (particularly iron overload disorders, even more particularly iron overload disorders characterized by ineffective erythropoiesis), wherein an effective amount of an anti-TMPRSS 6 antibody is administered such that more than one biological effect or symptom associated with the disorder is treated or alleviated. Without wishing to be bound by theory or mechanism of action, it is understood that ineffective erythropoiesis characterized by precursor apoptosis by the erythrocyte lines that result in very few mature erythrocytes in the bone marrow is the driving force for aberrant hepcidin repression leading to increased iron absorption and iron overload. In light of this understanding, treatments that improve erythroblasts differentiation and maturation into erythrocytes would be therapeutically beneficial in the treatment of iron overload conditions. The effectiveness of anti-TMPRSS 6 antibody therapy to increase erythrocyte differentiation and maturation into erythrocytes, reduce iron loading, increase hepcidin expression, etc., maximizes therapeutic benefit using the methods and compositions of anti-TMPRSS 6 antibodies disclosed herein.

Methods and compositions are provided for treating myeloproliferative disorders, particularly myeloproliferative neoplasms, such as chronic myeloproliferative neoplasms, more particularly myeloproliferative neoplasms characterized by erythrocyte proliferation, and even more particularly Polycythemia Vera (PV), wherein an effective amount of an anti-TMPRSS 6 antibody is administered such that more than one biological effect or symptom associated with the disorder is treated or ameliorated. Without wishing to be bound by theory or mechanism of action, in view of the degree of iron deficiency observed in PV patients, the observation of insufficiently suppressed hepcidin in PV patients is understood to indicate that disordered or deregulated iron metabolism is an important component of the PV pathology, and in particular, that the insufficiently suppressed hepcidin content is a component of the PV pathology. In light of this understanding, a treatment that regulates hepcidin expression should be therapeutically beneficial for the treatment of myeloproliferative neoplasms characterized by erythrocyte proliferation, in particular Polycythemia Vera (PV). anti-TMPRSS 6 antibody therapy reduces erythrocytosis and normalizes the blood volume ratio (HCT) level, and in particular increases the effectiveness of hepcidin expression to maximize therapeutic benefit using the methods and compositions of anti-TMPRSS 6 antibodies disclosed herein.

The following examples are provided for illustration, and are not intended to limit the claimed invention.

Examples

Example 1: antibody production and identification of antibodies that bind TMPRSS6

The generation of novel monoclonal antibodies against TMPRSS6 was performed under contract by the lycra pharmaceutical development immunology group (LAKEPHARMA DISCOVERY IMMUNOLOGY GROUP) (lycra pharmaceutical company (LAKEPHARMA, INC.SAN Carlos, CA) of san carbose, california) using in vivo rodent immunization and hybridoma technology. Using a mixture of pLEV113_ huTMPRSS6 and pLEV113_ moTMPRSS6-TCE plasmid DNA (cloned in lycra pharmaceutical company), at B6; DNA-based immunization was performed by fluid dynamic gene transfer tail intravenous injection in SJL mice (jackson laboratory (The Jackson Laboratories)). Sufficient plasma titers were obtained as determined by Fluorescence Activated Cell Sorting (FACS), triggering downstream antibody recovery and screening activities. The use of NEPA GENE ECFG super cell fusion generator (Nepa Gene, inc. (Nepa GeneCo., ltd., ichikawa-City, chiba, japan)) with 2 immunized mice and myeloma fusion partners pooled spleen cells were used for electrofusion. The fusion material was inoculated into hypoxanthine-aminopterin-thymidine medium in a total of ten (10) 384-well plates, which medium was specifically selected for fusion tumors within cells of the unfused myeloma partner. The hybridoma supernatants were first screened to measure HuTMPRSS for reactivity by FACS to detect positive staining signals on HEK293T cells expressing TMPRSS6 (plasmid encoding huTMPRSS- (His) ₆ (SEQ ID NO: 97) was transfected in HEK293T cells, HEK293T cells expressing TMPRSS6 were selected) and negative stained supernatants were generated on the parent (HEK 293T) on day 10 post-fusion. The hybridoma supernatants that produced positive staining signals on TMPRSS6 expressing HEK293 cells and negative staining on the parents were designated as "hit" for further screening. 192 hits were identified in the primary FACS screen and 143 hits were confirmed in the secondary and tertiary FACS screens.

Example 2 functional screening of anti-TMPRSS 6 antibodies; identification, generation and sequencing of monoclonal anti-TMPRSS 6 antibodies and humanized variants

HAMP-luciferase reporter assay

Hepcidin promoter-luciferase reporter assay was used to measure the response of the HAMP promoter to various anti-TMPRSS 6 antibodies (Du), x. Et al, 2008 Science 320:1088-1092; modified to use human HAMP promoter instead of the mouse HAMP promoter as originally disclosed). For the HAMP-luciferase reporter assay, a 2.5kb HAMP promoter fragment (reference genome GRCh 38) was spliced upstream from the sequence encoding firefly luciferase. A control construct encoding Renilla luciferase (Renilla luciferase) driven by the thymidine kinase promoter (Promega, E6931) was used as an internal control. These constructs were co-transfected into HepG2 cells (ATCC, HB-8065) along with the construct encoding TMPRSS 6. Transfected HepG2 cells expressing TMPRSS6 were pre-treated with purified mAb diluted in starvation medium containing minimum essential medium (MEM, ATCC) +1% heat inactivated fetal bovine serum (FBS, ji Buke (Gibco)) +1mM sodium pyruvate + nonessential amino acid solution (Ji Buke) +10mM HEPES (Ji Buke) +1% pen/Strep (Ji Buke) at various concentrations for about 3 hours, followed by treatment with recombinant hBMP6 (ani organisms (R & D Systems)) at final concentrations of 25-60ng/mL to trigger BMP-SMAD mediated signaling. Purified mouse IgG (Sigma-Aldrich) or human IgG1 (Bikei BioXcell) was used as a control. After overnight treatment of hBMP6, the cells were lysed and luciferase substrate was added. Luminescence readings from firefly luciferase and Renilla luciferase were each recorded by measuring total luminescence. The activity was calculated as the ratio of firefly luciferase luminescence to renilla luciferase luminescence (control). The results of these analyses are shown in fig. 2A to 2F.

In vitro functional screening

To screen functionally active hybridomas, the HAMP-luciferase reporter assay described above was used to test all 143 HuTMPRSS binding hybridomas ("hits"). The supernatant of ten (10) of 143 HuTMPRSS binding hybridomas increased HAMP promoter activity (data not shown) and were identified as "active clones" for further testing. These ten (10) active clones were tested for cross-reactivity against murine target MoTMPRSS6 as described in example 4 below, and three (3) showed binding to both HuTMPRSS and MoTMPRSS6 as measured by FACS. These three reactive cross clones were further seeded at a density of 1 cell/well into 192 wells of 384 well plates to generate monoclonal hybridoma clones, the resulting subclones identified as MWTx-001, MWTx-002, and MWTx-003, which exhibited the desired functional activity and cross-reactivity against non-human targets such as murine TMPRSS6 (moTMPRSS 6) and/or cynomolgus TMPRSS6 (cynoTMPRSS 6).

Sequences of anti-TMPRSS 6 antibodies MWTx-001, MWTx-002 and MWTx-003

MWTx-001, MWTx-002 and MWTx-003 were determined by: mRNA was isolated from each hybridoma sample and reverse transcription polymerase chain reaction (RT-PCR) was performed using a unique set of mouse IgG specific primers to amplify the variable region sequencing of interest. Unique heavy chains and unique light chains were identified for each anti-TMPRSS 6 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 heavy and light chain variable region amino acid sequences 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 heavy and light chain variable region nucleotide sequences.

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Generation and screening of humanized anti-TMPRSS 6 antibody variants

Humanization of the parent antibody is performed using CDR grafting onto the human antibody framework. Homology modeling of the 3-dimensional structure of the parent antibody is first performed to build a structural model of the parent antibody. Amino acid sequences of variable fragment frameworks were identified based on overall sequence identity, matching VH-VL interface positions, similarly classified CDR canonical positions, and removal of potential N-glycosylation sites. Humanized antibodies are designed by creating multiple hybrid sequences that fuse selected portions of the parent antibody sequence with human framework sequences. The isotype from which the humanized antibody was formed was selected to be IgG1 for the heavy chain and IgG1 kappa for the light chain. These humanized sequences were analyzed systematically by eye and computer modeling using 3D models to isolate sequences that would most likely retain antigen binding. The goal is to maximize the amount of human sequences in the final humanized antibody while maintaining the original antibody specificity. Humanized variants of paired humanized VH and VL were then expressed and purified for affinity analysis.

In one round of design, generation and testing of variants as part of affinity analysis, four VH variants were generated with VH-CDRs of parent antibody MWTX-003 in corresponding positions in four different human IgG 1-derived frameworks (SEQ ID NOs: 89-92) and four VL (VK) variants were generated with VL-CDRs of parent antibody MWTX-003 in corresponding positions in four different human IgG1 kappa-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 the 4VH x 4VK matrix, the antigen binding characteristics (k _on、k_off, KD) were evaluated and found to have KD values in the nanomolar range 4.16E-07 to 1.09E-08.

Variants exhibiting the desired antigen binding affinity were selected for further evaluation and development. In some cases, the parent CDR sequences are modified to avoid potentially undesirable events, such as aspartic acid isomerization.

To silence antibody effector function, in particular Antibody Dependent Cellular Cytotoxicity (ADCC), key amino acid residues in the Fc region are identified and mutated (substituted) for all humanized antibody variants. Guidance available in the publications on Fc mutations to achieve the goal of eliminating ADCC is used to understand the mutations of the invention, e.g. to remove the native Fc N-linked glycosylation site in hIgG1 (N297A mutation), or to replace leucine at positions 234 and 235 of the lower hinge region in Fc (LALA double mutation), as described in (Tamm) a, schmidt) RE. for IgG binding site on human fcγ receptor (IgG binding sites on human Fc gamma receptors); international immunoreview (Int Rev Immunol.) 1997;16 (1-2): 57-85.doi:10.3109/08830189709045703; jeffris (Jefferis) R, and the interaction site with fcγr on human IgG-Fc on Lund (Lund) j. Human IgG-Fc: current model (Interaction sites on human IgG-Fc for FcgammaR: current models); immunorapid report (Immunol lett.)) 2002 for 3 months; 82 (1-2): 57-65.doi: 10.3109/5278 (02-6). In variants of the invention, the N297A mutation was introduced into the Fc of hzMWTx-001Var and hzMWTx-002Var antibodies, and the LALA mutation was introduced into the Fc of hzMWTx-003Var antibodies to achieve the same objective of reducing or silencing ADCC (Table 3, SEQ ID NO:73, 77, 81).

After evaluation, humanized anti-TMPRSS 6 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.

Recombinant production of humanized anti-TMPRSS 6 antibody variants

The expression constructs of the humanized anti-TMPRSS 6 antibody variants were engineered to have internal ribosome entry sites (internal ribosome ENTRY SITE; IRES) between the LC-encoding and HC-encoding DNA sequences, codon-optimized by GENEART DNA synthesis and cloned into a pcDNA3.4 mammalian expression vector (ThermoFisher). The sequence of the DNA insert was verified by sequencing. For recombinant antibody production, expression constructs were used for transient transfection using ExpiCHO expression system (zemoeimeric) according to the manufacturer's instructions. The expressed antibodies were purified by protein a affinity chromatography. The yield of antibody produced by transient transfection ranged from 50mg to 300mg per liter with a purity >95% and <1EU/ml endotoxin content.

Humanized anti-TMPRSS 6 antibody variants hzMWTx-001Var, hzMWTx-002Var and hzMWTx-003Var sequences

Humanized anti-TMPRSS 6 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, wherein the identified CDRs are indicated by underlining and the changes in the humanized variant CDR sequences relative to the parent antibody are indicated and discussed.

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Table 3 shows the complete heavy and light chain proteins and nucleotide sequences of anti-TMPRSS 6 monoclonal antibodies MWTx-001, MWTx-002, and MWTx-003, as well as humanized anti-TMPRSS 6 antibody variants hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003 Var. The heavy chain protein sequences of humanized anti-TMPRSS 6 antibody variants hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var display mutated (altered) positions introduced as described above to reduce ADCC.

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Dose-dependent effect of anti-TMPRSS 6 antibodies on HAMP promoter activity

FIGS. 2A through 2F show the results of reporting assays using HAMP-luciferases described above to test MWTx-001, MWTx-002, MWTx-003 and humanized variants hzMWTx-001Var, hzMWTx-002Var, hzMWTx-003Var, respectively, at the indicated concentrations. Each of MWTx-001 (FIG. 2A), MWTx-002 (FIG. 2B), MWTx-003 (FIG. 2C), and the humanized variants hzMWTx-001Var (FIG. 2D), hzMWTx-002Var (FIG. 2E), hzMWTx-003Var (FIG. 2F) increased HAMP promoter activity in a dose-dependent manner. MWTx-001 EC ₅₀ was calculated to be 3 μg/ml (FIG. 2A). MWTx-002 EC ₅₀ was calculated to be 1 μg/ml (FIG. 2B). EC ₅₀ of MWTx-003 was calculated to be 2 μg/ml (FIG. 2C). EC ₅₀ of hzMWTx-001Var was calculated to be 0.8 μg/ml (FIG. 2D). EC ₅₀ of hzMWTx-002Var was calculated to be 0.3 μg/ml (FIG. 2E). EC ₅₀ of hzMWTx-003Var was calculated to be 0.3 μg/ml (FIG. 2F).

Example 3 binding affinity of anti-TMPRSS 6 antibodies

The binding affinity of various anti-TMPRSS 6 antibodies to human TMPRSS6 expressed on HEK293T cells was measured using the following three different methods: cell surface ELISA (fig. 3A to 3C), FACS (fig. 3D to 3F) and biological layer interferometry (fig. 3G to 3M).

Measurement of anti-TMPRSS 6 mAb binding affinity Using cell surface ELISA

HEK293T cells stably expressing human TMPRSS6 (produced by Lyck pharmaceutical Co., ltd., as described above; SEQ ID NO: 97) were fixed with 4% trioxymethylene (PFA) and washed with dPBS (Dulbecco's phosphate-buffered saline, corning cell gro) followed by incubation with various concentrations of anti-TMPRSS 6 antibodies diluted in BSA medium (DMEM+1% pen/strep+10mM HEPES+1mg/ml BSA (sigma aldrich)). Purified mouse IgG was used as a control (sigma aldrich). After incubation, cells were washed with BSA medium and then incubated with HRP-conjugated goat anti-mouse IgG as a2 ° antibody (Invitrogen). Finally, the cells were washed with pbs to remove unbound antibody and developed with ELISA liquid substrate (sigma aldrich), followed by stopping the reaction by adding the same volume of 1M H ₂SO₄ of ELISA liquid substrate. The bound antibody was measured by absorbance at OD _450nm. The results of these analyses are shown in figures 3A to 3C.

Anti-TMPRSS 6 mAb binding affinity measurement using FACS

HEK293T cells stably expressing human TMPRSS6 were collected and blocked with dps+3% bsa followed by incubation with various concentrations of anti-TMPRSS 6 antibodies diluted in dps+3% bsa. Purified mouse IgG was used as a control. After incubation, cells were washed with dPBS and then incubated with goat anti-mouse IgG as a 2 ° antibody (jackson immunoresearch company (Jackson ImmunoResearch Inc)) conjugated to APC. Finally, the cells were washed with dps to remove unbound antibody, resuspended with dps+1 mM EDTA, and then usedFACS analysis was performed with a flow cytometer (epa Biosciences, inc., san Diego CA). The bound antibodies were determined by measuring the average APC intensity after excitation at 640nm and measuring luminescence (fluorescence) at 675 nm. The results of these analyses are shown in figures 3D to 3F.

Anti-TMPRSS 6 antibody affinity and binding kinetics measurements using biolayer interferometry

Biological layer interferometry techniques for anti-TMPRSS 6 antibody affinity measurement and useBinding kinetics assay of RED96e System (Sartorius AG, germany). Prehydration anti-mouse IgG Fc capture (AMC) biosensors (for MWTx-001, MWTx-002, and MWTx-003 anti-TMPRSS 6 antibodies, FIGS. 3G through 3I) or anti-human IgG Fc capture (AHC) biosensors (for hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var anti-TMPRSS 6 antibodies, FIGS. 3J through 3L) were first equilibrated in 1 XKB (kinetic buffer, 1 XPBS pH7.4+0.02% Tween-20+0.1% BSA) for 120 seconds as a first baseline, followed by loading 10mg/mL anti-TMPRSS 6 antibody (MWTx-001, FIG. 3G; MWTx-002, FIG. 3H; MWTx-003, FIG. 3I; hzMWTx-001, FIG. 3J; hzMWTx-002Var, FIG. 3K; hzMWTx-003 seconds) onto the AMC or AHC biosensors and continuing 240 seconds. Next, a second baseline signal was established for 120 seconds before associating with various concentrations of human ecto-TMPRSS6-FLAG (SEQ ID NO: 102) (generated internally by fusing the extracellular domain of human TMPRSS6 with a FLAG-tag at the C-terminus) for 240 seconds. Finally, the analyte was dissociated in 1 XKB for 360 seconds. Data analysis was performed using eight-tuple data analysis HT software. KD. k _on、k_off and R ² are summarized in fig. 3M.

Example 4: cross-reactivity: anti-TMPRSS 6 antibodies that bind to human TMPRSS6 and non-human TMPRSS6

Determination of Cross-reactivity by FACS

The selected anti-TMPRSS 6 antibodies were tested to determine if any antibodies were able to bind to TMPRSS6 from mice and/or cynomolgus macaques. HEK293T cells stably expressing human TMPRSS6 (HuTMPRSS- (His) ₆) (produced by Lycra pharmaceutical Co., ltd., as described above), HEK293T cells stably expressing mouse TMPRSS6 (MoTMPRSS- (His) ₆) (SEQ ID NO: 98) (produced by Lycra pharmaceutical Co., ltd., as described above), and HEK293T cells transiently expressing cynomolgus monkey TMPRSS6 (CynoTMPRSS 6- (His) ₆) (SEQ ID NO: 99) (internal production) were collected. HEK293T cells stably expressing human TMPRSS6 served as positive controls and HEK293T cells served as negative controls (as described above). Cells were blocked with dps+3% bsa, followed by incubation with anti-TMPRSS 6 antibodies diluted in dps+3% bsa. After incubation, cells were washed with dPBS and then re-incubated with goat anti-mouse IgG conjugated to AlexaFluor-488 as the 2 ° antibody. Finally, the cells were washed with dps to remove unbound antibody, resuspended with dps+1 mM EDTA, and then usedFACS analysis was performed with a flow cytometer (eastern biosciences company, san diego, california). The bound antibody system was determined by excitation at 488nm and measurement of emission at 530nm (FITC-A). The results of these analyses are shown in the histograms in fig. 4A-4I. Cross-reactivity with mouse TMPRSS6 was observed for MWTx-001 (FIG. 4D) and MWTx-003 (FIG. 4F), whereas MWTx-002 (FIG. 4E) did not show detectable cross-reactivity with mouse TMPRSS 6. Cross-reactivity of MWTx-001 (FIG. 4G), MWTx-002 (FIG. 4H), and MWTx-003 (FIG. 4I) with cynomolgus monkey TMPRSS6 was observed.

Determination of Cross-reactivity by cell surface ELISA

HEK293T cells stably expressing mouse TMPRSS6 (produced by the lycra pharmaceutical company as described above, figures 4J, 4L, 4N, 4P, 4R, 4T) or HEK293T cells of cynomolgus monkey TMPRSS6 (produced internally as described above, figures 4K, 4M, 4O, 4Q, 4S, 4U) were fixed with methanol (100%) and washed with dPBS (dulbeck phosphate buffered saline, corning (Corning Cellgro)) followed by incubation with various concentrations of anti-TMPRSS 6 antibodies and humanized variants thereof diluted in BSA medium (dmem+1%pen/strep+10 mm hepes+1mg/ml BSA (sigma aldrich)). Purified mouse IgG (fig. 4J to 4O) or human IgG1 (fig. 4P to 4U) was used as a control. After incubation, cells were washed with BSA medium and then incubated with HRP-conjugated goat anti-mouse (invitrogen, fig. 4J to 4O) or anti-human (millbore, fig. 4P to 4U) IgG as a2 ° antibody. Finally, the cells were washed with pbs to remove unbound antibody and developed with ELISA liquid substrate (sigma aldrich), followed by stopping the reaction by adding the same volume of 1M H ₂SO₄ of ELISA liquid substrate. The bound antibody was measured by absorbance at OD _450nm. The results of these analyses are shown in figures 4J to 4U. Cross-reactivity of MWTx-001 (FIG. 4J) and MWTx-003 (FIG. 4N) anti-TMPRSS 6 antibodies and humanized variants thereof hzMWTx-001Var (FIG. 4P) and hzMWTx-003Var (FIG. 4T) anti-TMPRSS 6 antibodies with mouse TMPRSS6 was observed, whereas MWTx-002 (FIG. 4L) anti-TMPRSS 6 antibodies or humanized variants hzMWTx-002Var (FIG. 4R) anti-TMPRSS 6 antibodies did not exhibit detectable cross-reactivity with mouse TMPRSS 6. Cross-reactivity of MWTx-001 (FIG. 4K), MWTx-002 (FIG. 4M) and MWTx-003 (FIG. 4O) anti-TMPRSS 6 antibodies and humanized variants hzMWTx-001Var (FIG. 4Q), hzMWTx-002Var (FIG. 4S) and hzMWTx-003Var (FIG. 4U) anti-TMPRSS 6 antibodies with cynomolgus monkey TMPRSS6 were observed.

Example 5: target specificity: an anti-TMPRSS 6 antibody that binds to a cognate matrix protease.

To determine whether anti-TMPRSS 6 antibodies bind to cognate matrix proteases, HEK293T cells (FIGS. 5B, 5E, 5H, 5K, 5N, 5Q) overexpressing matrix protease (ST 14) (SEQ ID NO: 100) and HEK293T cells (FIGS. 5C, 5F, 5I, 5L, 5O, 5R) overexpressing matrix protease-3 (TMPRSS 7) (SEQ ID NO: 101) were collected (internally generated). HEK293T cells (produced by the lycra pharmaceutical company as described above, fig. 5A, 5D, 5G, 5J, 5M, 5P) stably expressing human TMPRSS6 (interstitial protease-2) (SEQ ID NO: 97) were used as positive controls and HEK293T cells (fig. 5A to 5R) were used as negative controls (as described above). Cells were blocked and permeabilized with dPBS+3% BSA+0.1% Tween-20, followed by incubation with various anti-TMPRSS 6 antibodies diluted in dPBS+3% BSA+0.1% Tween-20. Cells were incubated with anti-TMPRSS 6 antibodies and humanized variants thereof at a concentration of about 1 μg/ml for 1 hour. After incubation, cells were washed with dPBS and goat anti-mouse IgG conjugated to AlexaFluor-488 (England, FIGS. 5A through 5I) or goat anti-human IgG conjugated to allophycocyanin (Allophycocyanin; APC) (Jackson immune research Co (Jackson Immuno Research), FIGS. 5J through 5R) was incubated as a2℃antibody. Finally, the cells were washed with dPBS and resuspended with dpbs+1mM EDTA, and then usedFACS analysis was performed with a flow cytometer. The bound antibodies were determined by excitation at 488nm and measurement of luminescence (FITC-A) at 530nm (FIGS. 5A to 5I) or by excitation at 640nm and measurement of luminescence (APC-A) at 675nm (FIGS. 5J to 5R). The results of these analyses are shown in the histograms in fig. 5A to 5R. All antibodies displayed binding to human TMPRSS6 (interstitial protease-2) (fig. 5A, 5D, 5G, 5J, 5M, 5P) and none of the antibodies displayed binding to homologous interstitial protease ST14 (fig. 5B, 5E, 5H, 5K, 5N, 5Q) or TMPRSS7 (fig. 5C, 5F, 5I, 5L, 5O, 5R). MWTx-001 anti-TMPRSS 6 antibody and humanized variants thereof hzMWTx-001Var anti-TMPRSS 6 antibody displayed binding to human TMPRSS6 (fig. 5A, 5J) and did not display binding to either interstitial protease (ST 14) (fig. 5B, 5K) or interstitial protease-3 (TMPRSS 7) (fig. 5C, 5L). MWTx-002 anti-TMPRSS 6 antibodies and humanized variants thereof hzMWTx-002Var anti-TMPRSS 6 antibodies displayed binding to human TMPRSS6 (interstitial protease-2) (FIGS. 5D, 5M) and did not display binding to either interstitial protease (ST 14) (FIGS. 5E, 5N) or interstitial protease-3 (TMPRSS 7) (FIGS. 5F, 5O). MWTx-003 anti-TMPRSS 6 antibodies and humanized variants thereof hzMWTx-003Var anti-TMPRSS 6 antibodies displayed binding to human TMPRSS6 (interstitial protease-2) (FIGS. 5G, 5P) and did not display binding to either interstitial protease (ST 14) (FIGS. 5H, 5Q) or interstitial protease-3 (TMPRSS 7) (FIGS. 5I, 5R).

Example 6 treatment with anti-TMPRSS 6 antibodies in a mouse pharmacodynamic model

To study the in vivo pharmacodynamic response of anti-TMPRSS 6 antibodies, MWTx-003 anti-TMPRSS 6 antibodies (fig. 6A to 6B, 6D to 6E, 6G to 6H, 6J to 6K) or humanized variants thereof hzMWTx-003Var anti-TMPRSS 6 antibodies (fig. 6C, 6F, 6I, 6L) were injected intraperitoneally into wild-type C57BL/6J mice. Mouse IgG2B (bicaluri, fig. 6A to 6B, 6D to 6E, 6G to 6H, 6J to 6K) or human IgG1 (bicaluri, fig. 6C, 6F, 6I, 6L) was used as isotype control. 20 hours after injection, 50 μg of GFP-TMPRSS6 plasmid DNA (produced internally by insertion of human TMPRSS6 into GFP vector) was delivered into each mouse by hydrodynamic tail vein injection. Mice were euthanized 44 hours after hydrodynamic injection and liver tissue and blood were collected. Liver RNA was purified using EZgene Total RNA Purification Plus from bemisia (Biomiga) (san diego, california) according to the manufacturer's instructions. Mouse serum was obtained by centrifuging whole blood at 1500 Xg for 10min.

Effect of treatment with anti-TMPRSS 6 antibodies on serum iron

Serum iron was measured by an internally developed chromogenic assay (fig. 6A to 6C). Briefly, mouse serum or iron standard (31-500 μg/dL) was mixed with a mixed acid solution (0.6M trichloroacetic acid, 0.4M sodium thioglycolate, 1 MHCl) by vortexing for 30 seconds. The mixture was incubated at 37℃for 10min, then centrifuged at 10,000Xg for 10min, followed by development in a color solution (1.5M sodium acetate, 0.5mM bathophenanthroline disulfonate). The absorbance was then read at OD _535nm. Serum iron concentrations were calculated from linear iron standard curves. Treatment with 10mg/kg MWTx-003 anti-TMPRSS 6 antibody (fig. 6A-6B) and its humanized variants hzMWTx-003Var anti-TMPRSS 6 antibody (fig. 6C) significantly reduced serum iron.

Effects of treatment with anti-TMPRSS 6 antibodies on serum hepcidin

Serum hepcidin was measured by a hepcidin-mouse competition ELISA kit purchased from intrinsic life sciences (INTRINSIC LIFESCIENCES) (La Jolla, CA) according to the manufacturer's instructions (fig. 6D to 6F). Briefly, diluted mouse serum or hepcidin standard is mixed with hepcidin biotin conjugate and then added to the culture dish coated with anti-mouse hepcidin antibody. Serum hepcidin or hepcidin standards compete with hepcidin biotin conjugates for binding to coated anti-hepcidin antibodies. Bound hepcidin biotin conjugate was detected with streptavidin-bound horseradish peroxidase (HRP) and developed with TMB followed by stop solution development. The absorbance was then read at OD _450nm. Data were analyzed with a glafupand prism 8 (GRAPHPAD PRISM) using a four parameter logic (4-PL) curve fit and serum hepcidin concentrations were interpolated. Hydrodynamic delivery of GFP-TMPRSS6 significantly reduced serum hepcidin levels (fig. 6D), whereas treatment with 10mg/kg MWTx-003 anti-TMPRSS 6 antibody (fig. 6D to 6E) and its humanized variants hzMWTx-003Var anti-TMPRSS 6 antibody (fig. 6F) reversed hepcidin inhibition and significantly increased serum hepcidin levels.

Effect of treatment with anti-TMPRSS 6 antibody on hepcidin RNA

Hepcidin RNAs were quantified by real-time qPCR (fig. 6G to 6I). Briefly, cDNA was first synthesized from liver RNA using iScript reverse transcription super mix (Bio-Rad) according to the manufacturer's instructions. Hepcidin transcripts were amplified with specific primers listed below and SsoAdvanced ^TM Universal was used on a Bio-Rad CFX96 qPCR instrument according to manufacturer's instructionsGreen ultra-mix (Bio-Rad) was tested. Samples were analyzed in triplicate and the results were normalized to β -actin RNA content (measured by transcription, amplification with primers listed below, and quantification as described above). Hydrodynamic delivery of GFP-TMPRSS6 significantly reduced hepcidin RNA (fig. 6G). Treatment with 10mg/kg MWTx-003 anti-TMPRSS 6 antibody (FIGS. 6G through 6H) and its humanized variants hzMWTx-003Var anti-TMPRSS 6 antibody (FIG. 6I) reversed inhibition of Hamp and significantly increased hepcidin RNA content. The following primers were used for RNA quantification by real-time qPCR: hepcidin forward primer: 5'-AAG CAG GGC AGA CAT TGC GAT-3' (SEQ ID NO: 85); hepcidin reverse primer: 5'-CAG GAT GTG GCT CTA GGC TAT-3' (SEQ ID NO: 86); beta-actin forward primer: 5'-ACC CAC ACT GTG CCC ATC TA-3' (SEQ ID NO: 87); beta-actin reverse primer: 5'-CAC GCT CGG TCA GGA TCT TC-3' (SEQ ID NO: 88).

The serum concentration of MWTx-003 anti-TMPRSS 6 antibody or humanized variant hzMWTx-003Var anti-TMPRSS 6 antibody thereof was quantified by an internally developed cell surface ELISA (as described above, fig. 6J to 6L). Briefly, diluted mouse serum or anti-TMPRSS 6 antibody standard was incubated with 100% methanol-fixed HEK293T cells stably expressing human TMPRSS6 (HEK 293T cells were used as background control). Bound MWTx-003 anti-TMPRSS 6 antibody was detected with goat anti-mouse IgG bound to HRP, and bound hzMWTx-003Var anti-TMPRSS 6 antibody was detected with goat anti-human IgG bound to HRP. Color development with TMB followed by stop solution. The absorbance was then read at OD _450nm. Samples were analyzed in triplicate and the results were normalized to HEK293T control. Data were analyzed using four parameter logic (4-PL) curve fitting with glafupand prism 8 and serum anti-TMPRSS 6 antibody concentrations were interpolated.

Example 7 in vivo efficacy of anti-TMPRSS 6 antibodies using a mouse model of beta-thalassemia.

To investigate the in vivo efficacy of anti-TMPRSS 6 antibodies, a beta-thalassemia mouse model (B6.129P2-Hbb-b 1 ^tm1Unc Hbb-b2^tm1Unc/J, JAX Stock No:002683, jackson laboratories (The Jackson Laboratories), barbur, burma, referred to herein as Th3/+ mice) was selected. Th3/+ mice and their Wild Type (WT) littermates of 4 to 5 weeks of age were given a sufficient iron diet (Teklad TD.80394) and Th3/+ mice were treated every three days with 10mg/kg MWTx-003 anti-TMPRSS 6 antibody or mouse IgG2b isotype control for 4 weeks, while WT littermates did not receive treatment. At the end of the treatment process, mice were euthanized and spleen, liver, femur and blood samples were collected. Total liver RNA was purified and serum was collected as described above.

Effects on blood count, splenomegaly, serum iron, serum hepcidin, and hepcidin RNA

Whole blood cell count (CBC) was performed by VETSCAN HM automated hematology analyzer (FIGS. 7A-7D). MWTx-003 anti-TMPRSS 6 antibody treatment significantly increased the red blood cell count (RBC, fig. 7A) and the hematocrit (HCT, fig. 7C) and decreased the red blood cell distribution width (RDW, fig. 7D), but had no significant effect on heme (HGB, fig. 7B) in Th3/+ mice.

Spleen weights were measured, and MWTx-003 anti-TMPRSS 6 antibody treatment significantly reduced splenomegaly in Th3/+ mice (fig. 7E).

Serum iron was measured as described above. Treatment with MWTx-003 anti-TMPRSS 6 antibody significantly reduced serum iron (fig. 7F). Liver non-protoheme iron was measured using a similar chromogenic assay (fig. 7G). Briefly, minced small liver tissue was dried overnight at 65 ℃ and then digested with mixed acid (3 m hcl,10% trichloroacetic acid) at 65 ℃ for 20 hours. Next, the digested supernatant was collected to develop color in a color solution (1.5M sodium acetate, 0.5mM bathophenanthroline disulfonate). The absorbance was then read at OD _535nm. Treatment with MWTx-003 anti-TMPRSS 6 antibody significantly reduced liver non-protoheme iron (fig. 7G).

Serum hepcidin was measured by hepcidin-mouse competition ELISA kit as described above. Treatment with MWTx-003 anti-TMPRSS 6 antibody significantly increased serum hepcidin (fig. 7H).

Hepcidin RNA was quantified by real-time qPCR as described above. Treatment with MWTx-003 anti-TMPRSS 6 antibody significantly increased hepcidin RNA (fig. 7I).

Serum concentrations of MWTx-003 anti-TMPRSS 6 antibodies were quantified by an internally developed cell surface ELISA as described above (fig. 7J).

Effects on erythropoiesis

To investigate the effect of MWTx-003 anti-TMPRSS 6 antibodies on erythropoiesis in Th3/+ mice, bone marrow was collected from the femur (see fig. 7K to 7M) and spleen cells were collected from the spleen (see fig. 7N to 7P) and analyzed. The collected cells were blocked with rat anti-mouse CD16/CD32 (BD Biosciences) for 15min, followed by staining on ice for 30min with rat anti-mouse TER119 conjugated to FITC (BD Biosciences) and rat anti-mouse CD44 conjugated to APC (England). Washed cells were stained with viability marker 7-AAD (BD Biosciences) on ice for 10min, followed by useFACS analysis was performed with a flow cytometer. Ter119 ⁺, 7-ADD-cells were selected and density maps (FSC-H) were plotted against cell size with anti-mouse CD 44. The graph is analyzed to identify cell types (clumps) and the abundance of each type (clump) is determined. The representative graphs in fig. 7K-7P show that, corresponding to successive stages in erythrocyte differentiation, four different cell clumps are distinguished from top to bottom and are identified as: basophils (cluster I), polychromatics erythroblasts (cluster II), orthochromates erythroblasts and coreless reticulocytes (cluster III) and mature erythrocytes (cluster IV). The percent (%) value for each cluster in the sample was calculated as a measure of the abundance of cell types in the cluster, as shown in fig. 7K-7P. The% value of each cell cluster (I), (II), (III), (IV) was calculated for each sample of each animal (bone marrow, spleen) during each treatment as follows: WT (untreated) n=9; disease model Th3/+ mice treated with IgG2b isotype control (th3+w/MoIgG 2 b), n=5; disease model Th3/+ mice treated with MWTx-003 anti-TMPRSS 6 antibody (th3+w/MWTx-003), n=7, and then the average was calculated. On average, after four weeks, in bone marrow cells, the population of basophils (I) displayed 7.58% (th3+w/MoIgG b) to 6.52% (th3+w/MWTx-003) shift (7.96% for WT), polychromatic erythroblasts (II) displayed 54.20% (th3+w/MoIgG 2 b) to 40.01% (th3+w/MWTx-003) shift (28.53% for WT), orthochromatic erythroblasts and coreless reticulocytes (III) displayed 24.06% (th3+w/MoIgG 2 b) to 29.73% (th3+w/MWTx-003) shift (26.67% for WT) and mature erythrocytes (IV) displayed 4.54% (th3+w/MoIgG b) to 16.44% shift (27.66% for WT). On average, after four weeks, in the spleen, the population of basophils (I) displayed 0.71% (th3+w/MoIgG b) to 0.91% (th3+w/MWTx-003) shift (0.46% for WT), polychromatic erythroblasts (II) displayed 45.76% (th3+w/MoIgG b) to 19.25% (th3+w/MWTx-003) shift (12.23% for WT), orthochromatic erythroblasts and coreless reticulocytes (III) displayed 31.16% (th3+w/MoIgG 2 b) to 28.72% (th3+w/MWTx-003) shift (8.67% for WT) and mature erythrocytes (IV) displayed 14.13% (th3+w/MoIgG b) to 44.38% (th3+w/MWTx-003) shift (72.17% for WT). These results for bone marrow are shown in the bar graph of fig. 7Q and those for spleen are shown in the bar graph of fig. 7R.

In Th3/+ mice, treatment with MWTx-003 anti-TMPRSS 6 antibodies improved ineffective erythropoiesis, where a significant proportion of erythroblasts differentiated and matured into erythrocytes.

Example 8 anti-TMPRSS 6 antibody epitope grouping

RED96e was used for epitope grouping of MWTx-001 (FIG. 8A), MWTx-002 (FIG. 8B), and MWTx-003 (FIG. 8C) anti-TMPRSS 6 antibodies. First, ecto-TMPRSS6-FLAG (as described above) was labeled with biotin by the biotin labeling kit (Abcam). The pre-hydrated Streptavidin (SA) biosensor was equilibrated in 1 XKB (as described above) for 60 seconds as the first baseline, followed by loading 10mg/mL of biotin-labeled ecto-TMPRSS6-FLAG onto the SA biosensor for 300 seconds. Next, a second baseline signal was established for 60 seconds followed by saturation with 50mg/mL of antibody (MWTx-001, FIG. 8A; MWTx-002, FIG. 8B; MWTx-003, FIG. 8C) in 1 XKB for 600 seconds. Finally, a third baseline signal was established for 60 seconds, followed by competition with either MWTx-001, MWTx-002, or MWTx-003 for 50 μg/mL in 1 XKB for 300 seconds. MWTx-001 anti-TMPRSS 6 antibody bound to ecto-TMPRSS6-FLAG did not compete with MWTx-002 anti-TMPRSS 6 antibody or MWTx-003 anti-TMPRSS 6 antibody (FIG. 8A). MWTx-002 anti-TMPRSS 6 antibody bound to ecto-TMPRSS6-FLAG did not compete with MWTx-001 anti-TMPRSS 6 antibody, but did compete with MWTx-003 anti-TMPRSS 6 antibody (FIG. 8B). MWTx-003 anti-TMPRSS 6 antibody, which bound to ecto-TMPRSS6-FLAG, did not compete with MWTx-001 anti-TMPRSS 6 antibody, but did compete with MWTx-002 anti-TMPRSS 6 antibody (FIG. 8C). Data analysis was performed using eight-tuple data analysis HT software. The association signal is summarized in fig. 8D.

EXAMPLE 9 efficacy study of anti-TMPRSS 6 monoclonal antibodies in mouse models of polycythemia vera

The effect of anti-TMPRSS 6 recombinant monoclonal antibody treatment on reversing polycythemia and normalizing blood volume ratio levels in a murine model of Polycythemia Vera (PV) was evaluated.

B6n.129s6 (SJL) -Jak2 ^tm1.1Ble/AmlyJ mice (jax# 031658), commonly referred to as Jak2 ^V617F-Fl/+, are conditional gene knockout (floxed) lines with the reverse V617F mutation carrying exon 14 downstream of the endogenous exon 14 of the Janus kinase 2 (Jak 2) gene. The V617F mutation is common in patients with myeloproliferative neoplasms and is present in about 95% of PV patients. When breeding mice expressing tissue-specific Cre recombinase, the resulting offspring will remove the endogenous exon 14 of the conditional gene knockout and place the V617F mutant exon 14 in the correct transcription direction.

B6.Cg-Commd10 ^{Tg(Vav1-icre)A2Kio}/J mouse (JAX#008610), commonly referred to as Vav-iCre, specifically expresses an optimized variant of Cre recombinase (iCre) in hematopoietic cells and is suitable for generating conditional mutations in hematopoietic progenitor cells chambers. Offspring of Jak2 ^V617F-Fl/+ mice crossed with the Vav-iCre transgene can suffer from PV characterized by polycythemia and elevated blood volume ratio levels, and phenotypes can be propagated by transplanting bone marrow cells of double transgenic mice into lethally irradiated wild-type recipient mice.

Recombinant mouse anti-TMPRSS 6 monoclonal antibody MWTx-003, designated r4K12B in this study, was a recombinant expression version of mouse monoclonal MWTx-003, and may be referred to as recombinant monoclonal antibody MWTx-003, recombinant MWTx-003, or MWT-003 as in FIGS. 9A-9H. This in vivo repeat dose study of recombinant mouse anti-TMPRSS 6 monoclonal antibody r4K12B (the mouse counterpart of humanized antibody hzMWTx-003 Var) was used in a mouse PV model to avoid potential immunogenicity and generation of anti-drug antibodies (ADA). The recombinant mouse anti-TMPRSS 6 monoclonal antibody r4K12B has HC of SEQ ID NO:69 (HC amino acid sequence of mouse monoclonal MWTx-003) and LC of SEQ ID NO:71 (LC amino acid sequence of mouse monoclonal MWTx-003), is expressed by a vector in which the nucleotide of SEQ ID NO:70 (HC coding sequence of MWTx-003) and the nucleotide of SEQ ID NO:72 (LC coding sequence of MWTx-003) are inserted in a single vector having an engineered IRES between HC and LC coding sequences, and the expressed polypeptide is purified.

Material

The following materials were used to evaluate the effect of anti-TMPRSS 6 antibody treatment on reversing polycythemia and normalizing blood volume ratio levels in a mouse model of polycythemia vera.

R4K12B, recombinant mouse monoclonal antibody (recombinant MWTx-003), produced internally

A. Isotype: mouse IgG2b, kappa

B. batch: LN211201

C. concentration: 3.8mg/mL in PBS, pH 7.4

D. Purity: 95% by SDS-PAGE

E. endotoxin: 0.71EU/mg

InVivoPlus mouse IgG2b isotype control, purchased from biological cell company (BioXcell) (#BP 0086)

F. clone strain: MPC-11

G. batch: 779420O1

H. concentration: 10.26mg/mL in PBS, pH 7.0

I. purity: 95% by SDS-PAGE

J. Endotoxin: <1EU/mg

Method of

Animal study

Wild-type C57BL/6J (JAX # 000664) male mice of 10 to 12 weeks of age were purchased from Jackson laboratory (The Jackson Laboratory) and were acclimatized to the housing environment before the study began. All mice received a lethal dose of 1000cGy of whole body irradiation at 3.45 Gy/min. After 24 hours, 5×10 ⁶ bone marrow cells isolated from Jak2 ^V617/+ Vav-iCre double transgenic mice (using male and female mice) were injected via lateral tail veins into each lethally irradiated recipient C57BL/6J mouse. Immediately after Bone Marrow Transplantation (BMT), acidified drinking water (pH 2.5 to 3.0) containing antibiotics (sulfamethoxazole (sulfamethoxazole) and trimethoprim (trimethoprim)) was administered ad libitum for two weeks. The development of the PV phenotype of BMT animals was monitored by whole blood cell counting using an automated hematology analyzer. Four weeks after BMT, when the PV phenotype was fully established, mice received intraperitoneal injections of anti-TMPRSS 6 antibody r4K12B (recombinant MWTx-003) or mouse IgG2B isotype control antibody, once every 4 days for a total of 3 weeks. Animals were euthanized 4 days after the final dose, and bone marrow, spleen, liver, and whole blood were collected for analysis. The effect of anti-TMPRSS 6 antibody treatment on the red blood cell distribution, hematologic parameters (including mean red blood cell volume (MCV) and mean RBC size), splenomegaly, and tissue iron deposition of mice was evaluated.

Serum hepcidin, iron concentration and tissue iron deposition

Serum hepcidin was measured by hepcidin-mouse Compete ^TM ELISA (intrinsic life sciences (INTRINSIC LIFESCIENCES), sku#hmc-001) according to manufacturer's instructions as described above. The results are shown (fig. 9E).

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

Iron deposition in spleen and liver was assessed by Perls Prussian blue staining of 10% fumarin fixed liver and spleen sections. Slicing, staining and imaging work was contracted to ravigneaux biotechnology limited (Reveal Biosciences) (san diego, california). (FIG. 9H)

Analysis of hematology parameters

The erythrocyte index was analyzed by performing a whole blood count (CBC) on an HM5 VetScan hematology analyzer. (FIGS. 9A to 9C)

Erythroblasts were assessed for differentiation in bone marrow and spleen, respectively. Bone marrow collected from the femur and spleen cells from the spleen was analyzed by FACS as described above. The results are shown in (fig. 9G).

Measurement of anti-TMPRSS 6 antibody concentration in mouse serum

Serum concentrations of r4K12B anti-TMPRSS 6 antibody (recombinant MWTx-003) were quantified by internal cell surface ELISA as described above. The results are shown (fig. 9F).

Statistical analysis

Single factor ANOVA was used to compare three or more data sets using GRAPHPAD PRISM software. P <0.05 was considered statistically significant.

Results

Group allocation of herds

The body weight of bone marrow recipient C57BL/6J mice (all males) was measured during the adaptive randomization period to obtain similar average body weights between groups. Group assignment was performed according to the following table (table 4).

Table 4. Experimental group assignments.

Development of the PV phenotype in mice receiving Jak2 ^V617/+ Vav-iCre bone marrow cells

Blood samples were collected from recipient mice (wild-type C57BL/6J mice receiving Bone Marrow Transplantation (BMT) of Jak2V617/+ Vav-iCre bone marrow cells) and analyzed for hematological parameters 3-and 4-weeks after BMT, respectively. Reference Jak2 ^V617/+ Vav-iCre double transgenic mice (designated "PV reference line") the PV phenotype was produced in recipient mice 3 weeks after BMT and was fully established 4 weeks after BMT (table 5).

Table 5. Hematology parameters in lethally irradiated C57BL/6J recipients following bmt.

Administration of anti-TMPRSS 6 antibodies reversed erythrocytosis and normalized blood volume ratio levels in Jak2 ^V617/+ mouse PV models

4 Weeks after BMT, at which point the PV phenotype is fully established, mice (indicated as "PV phenotype" mice) receive intraperitoneal injections of 2mg/kg, 5mg/kg and 10mg/kg dose levels of anti-TMPRSS 6 antibody r4K12B (recombinant MWTx-003), or 10mg/kg mouse IgG2B isotype control, once every 4 days, for 3 weeks, respectively. Endpoint analysis was performed 4 days after the final injection.

After 2 weeks of treatment with anti-TMPRSS 6 antibody r4K12B, a dose-dependent decrease trend of the blood volume ratio (HCT) level, red Blood Cell (RBC) count and Heme (HGB) concentration was observed in mice receiving r4K12B compared to animals treated with isotype control antibody (table 6).

Table 6 hematological parameters in mice receiving anti-TMPRSS 6 antibodies for 2 weeks.

Results are expressed as mean ± SD, P <0.001, P <0.01, P <0.05, as compared to mIgG2b isotype control, using one-way ANOVA and danniter multiple comparison modulation, n=6 for mIgG2b group, n=8 for 10mg/kg group, n=7 for 5mg/kg and 2mg/kg group.

Results after 3 weeks of treatment

Fig. 9A-9C show endpoint measurements of hematology parameters HCT (fig. 9A), RBC (fig. 9B), and HGB (fig. 9C) for each treatment and dose level. Fig. 9D to 9E also show end point measurements for each treatment and dose level, where fig. 9D shows splenomegaly (splenomegaly index measured in mg/g body weight), fig. 9E shows serum hepcidin content (ng/ml), and fig. 9F shows serum anti-TMPRSS 6 concentration (μg/ml) measured by cell surface ELISA. Fig. 9G shows FACS results of measuring early erythrocyte precursors (I cluster basophils and II cluster polychromic erythroblasts) in bone marrow (upper row) and spleen (lower row), showing the results of WT (upper and lower row left panels), moIgG B isotype control (upper and lower row middle panels) and 10mg/kg anti-TMPRSS 6 r4K12B (MWTx-003) treatment (upper and lower row right panels). Fig. 9H shows liver (left panel) and spleen (right panel) sections stained to show iron content. In FIGS. 9A through 9H, labeled MWTx-003 indicates that antibody r4K12B was treated or measured with antibody r4K12B.

At the end of the 3-week treatment period, the HCT content in the r4k12B treated group was further reduced in a dose-dependent manner to levels similar to or lower than those seen in wild-type (WT) untreated animals (fig. 9A). The circulating RBC numbers and HGB concentrations were also reduced, with significantly greater reduction in erythrocytosis in the 10mg/kg dose group (fig. 9B-9C). Splenomegaly (fig. 9D) and early erythrocyte progenitor expansion (i.e., cluster I basophils and cluster II polychromic erythrocytes) were also observed in the 10mg/kg dose group (fig. 9G), indicating the development of iron-restricted erythropoiesis. As expected, serum hepcidin was significantly elevated and continued during the treatment (fig. 9E), resulting in a substantial decrease in serum iron concentration below that detected by colorimetric analysis (data not shown). These observations indicate that while the anti-TMPRSS 6 antibody r4K12B (MWTx-003) was effective 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. Fig. 9G shows representative FACS results of measuring early erythrocyte precursors in bone marrow (upper row) and spleen (lower row), wherein cluster I shows basophils and cluster II shows polychromic erythroblasts, which show the results of WT (left panel of upper and lower row), moIgG B isotype control (middle panel of upper and lower row), and 10mg/kg anti-TMPRSS 6 r4K12B (MWTx-003) treatment (right panel of upper and lower row). The sum percentages of cluster I and cluster II erythrocyte progenitors in the spleens in the wild type control, 10mg/kg moIgG B, and 10mg/kg r4K12B groups were 22.17.+ -. 1.74, 24.09.+ -. 4.52, and 40.06.+ -. 10.04, respectively. The% of the sum of the I and II clusters in r4K12B group was statistically different from the sum of moIgG B group and wild-type control mice (p= 0.0399 and p=0.0277, respectively), whereas there was no statistical difference between wild-type and moIgG B treated groups.

Fig. 9H shows Perls Prussian blue staining from control animals treated with mouse IgG2B isotype control MoIgG B (upper row) and animals treated with increasing doses of anti-TMPRSS 6 r4K12B (labeled MWTx-003) as indicated for fixed liver sections (left panel) and spleen sections (right panel) to measure iron deposits. The results in fig. 9H demonstrate that administration of the anti-TMPRSS 6 antibody r4K12B (MWTx-003) did not cause a significant change in liver iron content, but caused a significant increase in iron deposition in spleen macrophages, which increase was observed in a dose-dependent manner.

Conclusion(s)

Sub-chronic treatment with anti-TMPRSS 6 antibodies substantially reduced erythrocytosis and normalized hematocrit levels in a mouse model of polycythemia vera by limiting the iron available for erythrocyte precursors. anti-TMRSS antibody treatment provides a promising therapeutic pathway in PV management, where polycythemia and high HCT content are associated with poor results.

Claims

1. Use of an anti-TMPRSS 6 antibody to manufacture a medicament for treating a myeloproliferative disorder in a subject in need thereof, wherein the anti-TMPRSS 6 antibody comprises an antibody or antigen-binding fragment thereof that specifically binds to human TMPRSS6 and increases the activity of an hepcidin promoter, wherein the antibody is capable of binding to human TMPRSS6 on the surface of a cell expressing human TMPRSS6 and comprises one of:

i. (a) A heavy chain polypeptide comprising a heavy chain complementarity determining region 1HC CDR1 having sequence GYTFTSYW set forth in SEQ ID No. 2, HCCDR2 having sequence IYPGSGST set forth in SEQ ID No. 3, HC CDR3 having sequence APYDSDYAMDY set forth in SEQ ID No. 4; and (b) a light chain polypeptide comprising light chain complementarity determining region 1 (LC CDR 1) having sequence QDINNY set forth in SEQ ID No. 7, LC CDR2 having sequence RAN set forth in SEQ ID No. 8, LC CDR3 having sequence LQYDEFPLT set forth in SEQ ID No. 9;

(a) a heavy chain polypeptide comprising HCCDR1 having sequence GYTFTSYW set forth in SEQ ID No. 32, HC CDR2 having sequence IYPGSGST set forth in SEQ ID No. 33, HC CDR3 having sequence APYDADYAMDY set forth in SEQ ID No. 34; and (b) a light chain polypeptide comprising LC CDR1 having sequence QDISNY of SEQ ID No. 37, LC CDR2 having sequence RAN set forth in SEQ ID No. 38, LC CDR3 having sequence LQYDEFPLT set forth in SEQ ID No. 39;

(a) a heavy chain polypeptide comprising HCCDR1 having sequence GFNIKDYY set forth in SEQ ID No. 12, HC CDR2 having sequence IDPEDGES set forth in SEQ ID No. 13, HC CDR3 having sequence TRGDSMMVTYFDY set forth in SEQ ID No. 14; and (b) a light chain polypeptide comprising LC CDR1 having sequence QDVSTA set forth in SEQ ID No. 17, LC CDR2 having sequence WAF set forth in SEQ ID No. 18, LC CDR3 having sequence QQHYRSPWT set forth in SEQ ID No. 19;

(a) a heavy chain polypeptide comprising HCCDR1 having sequence GFNIKDYY set forth in SEQ ID No. 42, HC CDR2 having sequence IDPEDAES set forth in SEQ ID No. 43, HC CDR3 having sequence TRGDSMMVTYFDY set forth in SEQ ID No. 44; and (b) a light chain polypeptide comprising LC CDR1 having sequence QDVSTA set forth in SEQ ID No. 47, LC CDR2 having sequence WAF set forth in SEQ ID No. 48, LC CDR3 having sequence QQHYRSPWT set forth in SEQ ID No. 49;

(a) a heavy chain polypeptide comprising HCCDR1 having sequence GFNIEDYY set forth in SEQ ID No. 22, HC CDR2 having sequence IDPEDGET set forth in SEQ ID No. 23, HC CDR3 having sequence ARSIYLDPMDY set forth in SEQ ID No. 24; and (b) a light chain polypeptide comprising LC CDR1 having sequence QDVTTA set forth in SEQ ID No. 27 or SEQ ID No. 57, LC CDR2 having sequence WAT set forth in SEQ ID No. 58, LC CDR3 having sequence QQHYSTPYT set forth in SEQ ID No. 29; or (b)

(A) a heavy chain polypeptide comprising HCCDR1 having sequence GFNIEDYY set forth in SEQ ID No. 52, HC CDR2 having sequence IDPEDAET set forth in SEQ ID No. 53, HC CDR3 having sequence ARSIYLDPMDY set forth in SEQ ID No. 54; and (b) a light chain polypeptide comprising LC CDR1 having sequence QDVTTA set forth in SEQ ID No. 57, LC CDR2 having sequence WAT set forth in SEQ ID No. 58, LC CDR3 having sequence QQHYSTPYT set forth in SEQ ID No. 59.

2. The use of claim 1, wherein the myeloproliferative disorder is a myeloproliferative neoplasm.

3. The use of claim 2, wherein the myeloproliferative neoplasm is polycythemia vera PV.

4. The use of claim 1, wherein the anti-TMPRSS 6 antibody has at least one effect selected from the group consisting of: reducing red blood cell count RBC, reducing Hematocrit (HCT), reducing Heme (HGB), reducing mean red blood cell volume MCV, and reducing red blood cell distribution width RDW.

5. The use of claim 3, wherein the anti-TMPRSS 6 antibody has at least one effect selected from the group consisting of: reducing red blood cell count RBC, reducing Hematocrit (HCT), reducing Heme (HGB), reducing mean red blood cell volume MCV, and reducing red blood cell distribution width RDW.

6. The use of claim 1, wherein the anti-TMPRSS 6 antibody is selected from at least one of the following: monoclonal antibodies, chimeric antibodies, humanized antibodies, recombinant antibodies, and antigen-binding fragments.

7. The use of claim 1, wherein the medicament further comprises a pharmaceutically acceptable carrier.

8. The use of claim 1, wherein the individual is a human.

9. The use of claim 1, wherein the anti-TMPRSS 6 antibody comprises at least one polypeptide having an amino acid sequence at least about 85%, 90%, 92%, 95%, 97% or 98%, 99% or 100% identical to 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; the sequence WAF set forth in SEQ ID NO. 8 as set forth in SEQ ID NO. RAN;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; sequence WAT;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;SEQ ID NO:73;SEQ ID NO:75;SEQ ID NO:77;SEQ ID NO:79;SEQ ID NO:81; as set forth in sequence WAF;SEQ ID NO:49;SEQ ID NO:51;SEQ ID NO:52;SEQ ID NO:53;SEQ ID NO:54;SEQ ID NO:56;SEQ ID NO:57;SEQ ID NO:58 as set forth in sequence RAN;SEQ ID NO:39;SEQ ID NO:41;SEQID NO:42;SEQ ID NO:43;SEQ ID NO:44;SEQ ID NO:46;SEQ ID NO:47;SEQID NO:48 as set forth in sequence WAT;SEQ ID NO:29;SEQ ID NO:31;SEQ ID NO:32;SEQ ID NO:33;SEQ ID NO:34;SEQ ID NO:36;SEQ ID NO:37;SEQ ID NO:38 as set forth in SEQ ID NO. 28 or SEQ ID NO. 83.

10. The use of claim 1, wherein the anti-TMPRSS 6 antibody comprises one of:

a. (a) A heavy chain HC polypeptide, wherein the variable region comprises an amino acid sequence that is at least about 85%, 90%, 92%, 95%, 97% or 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO. 1; and (b) a light chain LC polypeptide, wherein the variable region comprises an amino acid sequence that is at least about 85%, 90%, 92%, 95%, 97% or 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 6;

b. (a) A HC polypeptide, wherein the variable region comprises an amino acid sequence that is at least about 85%, 90%, 92%, 95%, 97% or 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO. 31; and (b) a light chain LC polypeptide, wherein the variable region comprises an amino acid sequence that is at least about 85%, 90%, 92%, 95%, 97% or 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 36;

c. (a) A HC polypeptide, wherein the variable region comprises an amino acid sequence that is at least about 85%, 90%, 92%, 95%, 97% or 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO. 11; and (b) an LC polypeptide, wherein the variable region comprises an amino acid sequence that is at least about 85%, 90%, 92%, 95%, 97% or 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 16;

d. (a) A HC polypeptide, wherein the variable region comprises an amino acid sequence that is at least about 85%, 90%, 92%, 95%, 97% or 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO. 41; and (b) an LC polypeptide, wherein the variable region comprises an amino acid sequence at least about 85%, 90%, 92%, 95%, 97% or 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO. 46;

e. (a) A HC polypeptide, wherein the variable region comprises an amino acid sequence that is at least about 85%, 90%, 92%, 95%, 97% or 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO. 21; and (b) an LC polypeptide, wherein the variable region comprises an amino acid sequence at least about 85%, 90%, 92%, 95%, 97% or 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO. 26; or (b)

F. (a) A HC polypeptide, wherein the variable region comprises an amino acid sequence that is at least about 85%, 90%, 92%, 95%, 97% or 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO. 51; and (b) a light chain LC polypeptide, wherein the variable region comprises an amino acid sequence that is at least about 85%, 90%, 92%, 95%, 97% or 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 56.

11. The use of claim 1, wherein the anti-TMPRSS 6 antibody comprises:

(a) A heavy chain polypeptide comprising HCCDR1 having sequence GFNIEDYY set forth in SEQ ID NO. 22, HC CDR2 having sequence IDPEDGET set forth in SEQ ID NO. 23, HC CDR3 having sequence ARSIYLDPMDY set forth in SEQ ID NO. 24, and

(B) A light chain polypeptide comprising LC CDR1 having sequence QDVTTA set forth in SEQ ID No. 27 or SEQ ID No. 57, LC CDR2 having sequence WAT set forth in SEQ ID No. 58, LC CDR3 having sequence QQHYSTPYT set forth in SEQ ID No. 29.

12. The use of claim 1, wherein the anti-TMPRSS 6 antibody comprises:

(a) A heavy chain polypeptide comprising HCCDR1 having sequence GFNIEDYY set forth in SEQ ID NO:52, HC CDR2 having sequence IDPEDAET set forth in SEQ ID NO:53, HC CDR3 having sequence ARSIYLDPMDY set forth in SEQ ID NO:54, and

(B) A light chain polypeptide comprising LC CDR1 having sequence QDVTTA set forth in SEQ ID No. 57, LC CDR2 having sequence WAT set forth in SEQ ID No. 58, LC CDR3 having sequence QQHYSTPYT set forth in SEQ ID No. 59.

13. The use of claim 10, wherein the anti-TMPRSS 6 antibody comprises:

(a) HC polypeptide wherein the variable region comprises an amino acid sequence at least about 85%, 90%, 92%, 95%, 97% or 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO. 21 and

(B) An LC polypeptide, wherein the variable region comprises an amino acid sequence at least about 85%, 90%, 92%, 95%, 97% or 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 26.

14. The use of claim 10, wherein the anti-TMPRSS 6 antibody comprises:

(a) HC polypeptide wherein the variable region comprises an amino acid sequence at least about 85%, 90%, 92%, 95%, 97% or 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO. 51 and

(B) A light chain LC polypeptide, wherein the variable region comprises an amino acid sequence that is at least about 85%, 90%, 92%, 95%, 97% or 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 56.