Anti-TMPRSS6 antibody and its uses

Abstract

This provides a treatment method for iron metabolism disorders using antibodies. [Solution] Provided are an antibody and its antigen-binding fragment that bind to type II transmembrane serine protease 6 (TMPRSS6) on the cell surface and increase hepcidin expression, as well as a method for treating iron metabolism disorders using the anti-TMPRSS6 antibody and fragment. The anti-TMPRSS6 antibody disclosed herein can be used to treat iron overload, particularly iron metabolism disorders such as β-thalassemia including non-transfusion-dependent thalassemia, and other disorders of ineffective hematopoiesis.

Description

Related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 006,695, entitled “Anti-TMPRSS6 Antibodies and Uses Thereof,” filed on 7 April 2020, and U.S. Provisional Application No. 63 / 158,265, entitled “Anti-TMPRSS6 Antibodies and Uses Thereof,” filed on 8 March 2021, the entire contents of which are incorporated herein by reference. [Sequence List]

[0002] This application includes an electronically submitted sequence listing in ASCII format, the entire listing of which is incorporated herein by reference. The ASCII copy, created on March 29, 2021, is named 1121_101PCT_SL.txt and has a size of 132,677 bytes. [Technical Field]

[0003] This disclosure relates to antibodies and antigen-binding fragments that conjugate to TMPRSS6, and to the treatment of iron metabolism disorders using antibodies and antigen-binding fragments that conjugate to TMPRSS6. [Background technology]

[0004] Type II transmembrane serine protease 6 (TMPRSS6) is encoded by the TMPRSS6 gene and is mainly expressed in the liver. The structure of TMPRSS6 includes a type II transmembrane domain, a stem region containing sea urchin sperm protein, enteropeptidase and agrin (SEA) domains, two complement factor C1r / C1s, sea urchin embryo growth factor and bone morphogenetic protein (CUB) domains, and three low-density lipoprotein receptor (LDLR) class A repeats, as well as a C-terminal trypsin-like serine protease domain (Wang, C.-Y. et al., Front. Pharmacol). .(2014.5:114). Other names for TMPRSS6 (EC3.4.21) include matryptase-2; transmembrane protease serine 6; membrane-bound mosaic serine proteinase matryptase-2; and MT2.

[0005] TMPRSS6 plays a crucial role in iron homeostasis through the BMP-SMAD signaling pathway, which controls the expression of hepcidin, a hormone that regulates iron absorption and transport from stored iron. Hepcidin (also known as HAMP (hepcidin anti-microbial 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 ferroportin, the sole iron efflux channel. Hepcidin can lower plasma iron concentration by binding to ferroportin and causing its internalization and degradation, thereby hindering iron absorption in the small intestine and the release of stored iron. Chronic elevated hepcidin levels lead to systemic iron deficiency, while hepcidin deficiency leads to systemic iron overload.

[0006] TMPRSS6 negatively regulates hepcidin production through a transmembrane signaling pathway induced by iron deficiency, thereby suppressing HAMP activation (Du, X. et al., Science 2008.320:1088-1092; Wang, C.-Y. et al., Front.Pharmacol). . (2014.5:114). A decrease in blood iron concentration activates this pathway, reducing hepcidin production. This allows more iron from the diet to be absorbed from the intestines and transported from storage sites into the bloodstream. In rats under acute iron deficiency, hepatic TMPRSS6 protein concentration increases, and hepcidin expression and production are suppressed (Wang, C.-Y. et al., Front. Pharmacol). .(2014.5:114). Mutations in the entire TMPRSS6 molecule, particularly in the extracellular domain, have been identified in patients with iron deficiency anemia, especially iron-refractory iron deficiency anemia (IRIDA) that does not respond to oral iron therapy and only partially responds to parenteral iron therapy (Wang, C.-Y. et al., Front.Pharmacol). . (2014.5:114). Loss-of-function mutations in human TMPRSS6 lead to excessive hepcidin production, which impairs iron absorption and utilization, resulting in elevated hepcidin levels and iron deficiency anemia (Camaschella, C., N Engl Journal Med 2013.168:24).

[0007] Iron overload occurs when excess iron accumulates in tissues and organs, impairing their normal function. Iron toxicity is a typical complication of iron overload, and the accumulation of iron in major organs leads to a high mortality rate. Beta-thalassemia is a disease in which mutations in the HBB gene reduce or eliminate the production of beta-globin, leading to apoptosis of erythroblasts and a deficiency of mature red blood cells. As a result, red blood cell production is impaired, leading to anemia and iron overload due to excessive iron absorption. In patients with beta-thalassemia, hepcidin is abnormally suppressed depending on the patient's iron load, resulting in hepcidin deficiency, excessive iron absorption, and the progression of systemic iron overload. Ineffective hematopoiesis, such as MDS (myelodysplastic syndrome), abnormal hematopoietic anemia, and sideroblastic anemia, is also characterized by iron overload due to decreased hepcidin levels. Hemochromatosis (including type 1 hemochromatosis and hereditary hemochromatosis) is an iron overload characterized by excessive absorption of dietary iron in the intestines and a pathological increase in systemic iron stores. Current standard treatments for iron overload include transfusions for ineffective hematopoiesis that may further worsen the condition, iron chelation with low patient compliance, and phlebotomy or splenectomy to manage symptoms. Therapeutic approaches currently under development include gene therapy targeting the HBB gene, gene therapy and gene editing targeting other related genes, hepcidin mimetic drugs, Fc fusion proteins that target TGF superfamily ligands to inhibit SMAD signaling, antisense RNA drugs targeting TMPRSS6 (e.g., El-Beshlawy A., et al., Blood Cells, Molecules and Diseases 2019.76:53-58), and iRNA drugs targeting TMPRSS6. [Overview of the project]

[0008] The present invention relates to a novel antibody that binds to TMPRSS6 and its antigen-binding fragment, as well as a method for producing and using the antibody that binds to TMPRSS6 and its antigen-binding fragment.

[0009] This disclosure provides anti-TMPRSS6 antibodies, nucleic acids encoding anti-TMPRSS6 antibodies, and methods for producing and using anti-TMPRSS6 antibodies. The anti-TMPRSS6 antibodies disclosed herein include anti-TMPRSS6 antibodies and fragments thereof that can bind to TMPRSS6. The anti-TMPRSS6 antibodies disclosed herein can bind to human TMPRSS6 on the surface of cells expressing human TMPRSS6. This disclosure provides anti-TMPRSS6 antibodies for therapeutic and diagnostic applications. The anti-TMPRSS6 antibodies disclosed herein can be used to treat iron overload, particularly β-thalassemia including non-transfusion-dependent thalassemia, and other disorders of iron metabolism such as ineffective hematopoiesis.

[0010] In one embodiment, an anti-TMPRSS6 antibody is provided that can bind to TMPRSS6 on the surface of cells expressing TMPRSS6 and modulate the activity of at least one component involved in iron metabolism, where the component may be a molecule or biological process related to the function of TMPRSS6. In certain embodiments, the anti-TMPRSS6 antibody disclosed herein can modulate the activity of at least one component involved in the regulation of hepcidin expression. In certain embodiments, the anti-TMPRSS6 antibody disclosed herein can substantially inhibit the TMPRSS6 repression of hepcidin expression. In certain embodiments, the anti-TMPRSS6 antibody disclosed herein can increase hepcidin expression. In certain embodiments, the anti-TMPRSS6 antibody disclosed herein can increase the activity of the hepcidin promoter. In certain embodiments, the anti-TMPRSS6 antibody disclosed herein can substantially inhibit the BMP / SMAD pathway-induced repression of hepcidin expression by TMPRSS6. The anti-TMPRSS6 antibodies disclosed herein, without limitation, can dose-dependently modulate hepcidin expression by substantially inhibiting TMPRSS6 repression of hepcidin expression, increasing hepcidin expression, increasing hepcidin promoter activity, or substantially inhibiting TMPRSS6-induced repression of hepcidin expression via the BMP / SMAD pathway. In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein can dose-dependently modulate hepcidin expression. In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein can dose-dependently increase serum hepcidin concentration when administered to a subject. In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein can dose-dependently decrease serum iron concentration when administered to a subject. In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein can dose-dependently increase hepatic hepcidin RNA concentration when administered to a subject.In certain embodiments, the anti-TMPRSS6 antibody disclosed herein, when administered to subjects known or suspected of having iron overload, particularly β-thalassemia, enables the reduction of hepatic non-heme iron, an increase in serum hepcidin, an increase in hepatic hepcidin RNA, a reduction in splenomegaly, an increase in red blood cell count (RBC), a decrease in hematocrit, a decrease in red blood cell distribution width (RDW), and an increase in the production of mature red blood cells (increased erythropoiesis).

[0011] In another embodiment, the anti-TMPRSS6 antibody disclosed herein exhibits cross-reactivity with at least one non-human TMPRSS6. In certain embodiments, the anti-TMPRSS6 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-TMPRSS6 antibody disclosed herein is capable of binding to human TMPRSS6 and mouse TMPRSS6. The anti-TMPRSS6 antibody disclosed herein is capable of binding to human TMPRSS6 and cynomolgus monkey TMPRSS6. The anti-TMPRSS6 antibody disclosed herein can bind to human TMPRSS6, mouse TMPRSS6, and cynomolgus monkey TMPRSS6, respectively.

[0012] In another embodiment, the anti-TMPRSS6 antibody disclosed herein specifically binds to TMPRSS6 (matryptase-2). In certain embodiments, the anti-TMPRSS6 antibody disclosed herein binds to TMPRSS6 (matryptase-2) and does not show detectable binding to other matryptase congeners. In certain embodiments, the anti-TMPRSS6 antibody disclosed herein binds to human TMPRSS6 (matryptase-2) and does not show detectable binding to human matryptase-1 (ST14). In certain embodiments, the anti-TMPRSS6 antibody disclosed herein binds to human TMPRSS6 (matryptase-2) and does not show detectable binding to human matryptase-3 (TMPRSS7). In certain embodiments, the anti-TMPRSS6 antibody disclosed herein binds to human TMPRSS6 (matryptase 2) and does not exhibit detectable binding to either human matryptase-1 (ST14) or human matryptase-3 (TMPRSS7).

[0013] The anti-TMPRSS6 antibodies disclosed herein may be monoclonal antibodies, humanized antibodies, chimeric antibodies, single-chain antibodies, Fab fragments, single-chain variable fragments (scFv), recombinant antibodies, aptamers, single-domain antibodies (VHH, nanobodies), or other TMPRSS6-binding fragments or variants. In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein may include frameworks in which amino acids have been substituted into an existing antibody framework, particularly to affect properties such as antigen-binding ability. In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein may include frameworks from an antibody of a different type (class) and / or from a different organism, particularly complementarity-determining regions (CDRs) from a source (parent) antibody grafted (fused) onto an acceptor human framework. In certain embodiments, the anti-TMPRSS6 antibody disclosed herein may include regions other than the CDR, for example, a variable region framework surrounding the CDR and / or a constant region, particularly the Fc region, in which amino acids are substituted, mutated, or replaced, in order to affect properties such as antigen binding or antibody structure. In certain embodiments, one or more CDRs are substituted, mutated, or replaced. In certain embodiments, the anti-TMPRSS6 antibody disclosed herein may be a humanized anti-TMPRSS6 antibody variant.

[0014] In certain embodiments, the anti-TMPRSS6 antibody disclosed herein comprises at least one polypeptide having an amino acid sequence listed in Table 1, Table 2, or Table 3, or a sequence substantially identical (e.g., at least 85%, 90%, 92%, 95%, 97%, or 98%, 99% identical) to an amino acid sequence listed in Table 1, Table 2, or Table 3. The anti-TMPRSS6 antibody disclosed herein may also comprise an amino acid sequence selected from the following, or a sequence substantially identical (e.g., at least 85%, 90%, 92%, 95%, 97%, or 98%, 99% identical) to at least one polypeptide having an amino acid sequence selected from the following. Array 1; Array 2; Array 3; Array 4; Array 6; Array 7; Array 8; Array 9; Array 11; Array 12; Array 13; Array 14; Array 16; Array 17; Array 18; Array 19; Array 21; Array 22; Array 23; Array 24; Array 26; Array 27; Array 28; Array 29; Array 31; Array 32; Array 33; Array 34; Array 36; Array 37; Array 38; Array 39; Array 41; Array 42; Array 43; Array 44; Array 46; Array 47; Array 48; Array 49; Array 51; Array 52; Array 53; Array 54; Array 56; Array 57; Array 58; Array 59; Array 61; Array 63; Array 65; Array 67; Array 69; Array 71; Array 73; Array 75; Array 77; Array 79; Array 81; or Array 83.

[0015] In one embodiment, the anti-TMPRSS6 antibody disclosed herein comprises a heavy chain (HC) variable region polypeptide having the amino acid sequence described in SEQ ID NO: 1, or a sequence substantially identical to that of SEQ ID NO: 1, and a light chain (LC) variable region polypeptide having the amino acid sequence described in SEQ ID NO: 6, or a sequence substantially identical to that of SEQ ID NO: 6. In one embodiment, the anti-TMPRSS6 antibody disclosed herein comprises a variant of the antibody comprising the heavy chain complementarity determination region 1 (HC CDR1) of the amino acid sequence described in SEQ ID NO: 2, the heavy chain complementarity determination region 2 (HC CDR2) of the amino acid sequence described in SEQ ID NO: 3, the heavy chain complementarity determination region 3 (HC CDR3) of the amino acid sequence described in SEQ ID NO: 4, the light chain complementarity determination region 1 (LC CDR1) of the amino acid sequence described in SEQ ID NO: 7, the light chain complementarity determination region 2 (LC CDR2) of the amino acid sequence described in SEQ ID NO: 8, and the light chain complementarity determination region 3 (LC CDR3) of the amino acid sequence described in SEQ ID NO: 9; or substitutions of 1, 2, 3, 4, 5, or 6 amino acids in the CDR region. In one non-limiting embodiment, the anti-TMPRSS6 antibody disclosed herein is an antibody identified herein as MWTx-001, comprising an HC polypeptide having the amino acid sequence described in SEQ ID NO: 61 and an LC polypeptide having the amino acid sequence described in SEQ ID NO: 63.

[0016] In one embodiment, the anti-TMPRSS6 antibody disclosed herein comprises a heavy chain (HC) variable region polypeptide having the amino acid sequence described in SEQ ID NO: 11, or a sequence substantially identical to that of SEQ ID NO: 11, and a light chain (LC) variable region polypeptide having the amino acid sequence described in SEQ ID NO: 16, or a sequence substantially identical to that of SEQ ID NO: 16. In one embodiment, the anti-TMPRSS6 antibody disclosed herein comprises an HC CDR1 having the amino acid sequence described in SEQ ID NO: 12, an HC CDR2 having the amino acid sequence described in SEQ ID NO: 13, an HC CDR3 having the amino acid sequence described in SEQ ID NO: 14, an LC CDR1 having the amino acid sequence described in SEQ ID NO: 17, an LC CDR2 having the amino acid sequence described in SEQ ID NO: 18, and an LC CDR3 having the amino acid sequence described in SEQ ID NO: 19, or a variant of the antibody comprising substitutions of 1, 2, 3, 4, 5, or 6 amino acids in the CDR region. In one non-limiting embodiment, the anti-TMPRSS6 antibody disclosed herein is an antibody identified herein as MWTx-002, comprising an HC polypeptide having the amino acid sequence described in SEQ ID NO: 65 and an LC polypeptide having the amino acid sequence described in SEQ ID NO: 67.

[0017] In one embodiment, the anti-TMPRSS6 antibody disclosed in the present disclosure comprises a heavy chain (HC) variable region polypeptide having the amino acid sequence set forth in SEQ ID NO: 21 or a sequence substantially identical to SEQ ID NO: 21, and a light chain (LC) variable region polypeptide having the amino acid sequence set forth in SEQ ID NO: 26 or a sequence substantially identical to SEQ ID NO: 26. In one embodiment, the anti-TMPRSS6 antibody disclosed in the present disclosure comprises HC CDR1 having the amino acid sequence set forth in SEQ ID NO: 22, HC CDR2 having the amino acid sequence set forth in SEQ ID NO: 23, HC CDR3 having the amino acid sequence set forth in SEQ ID NO: 24, LC CDR1 having the amino acid sequence set forth in SEQ ID NO: 27, LC CDR2 having the amino acid sequence set forth in SEQ ID NO: 28, and LC CDR3 having the amino acid sequence set forth in SEQ ID NO: 29, or variants of said antibody comprising substitutions of 1, 2, 3, 4, 5 or 6 amino acids in the CDR regions. In one non-limiting embodiment, the anti-TMPRSS6 antibody disclosed in the present disclosure is an antibody identified as MWTx-003 in the present disclosure, comprising 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.

[0018] In one embodiment, the anti-TMPRSS6 antibody disclosed herein comprises a heavy chain (HC) variable region polypeptide having the amino acid sequence described in SEQ ID NO: 31, or a sequence substantially identical to that of SEQ ID NO: 31, and a light chain (LC) variable region polypeptide having the amino acid sequence described in SEQ ID NO: 36, or a sequence substantially identical to that of SEQ ID NO: 36. In one embodiment, the anti-TMPRSS6 antibody disclosed herein comprises a variant of the antibody having an HC CDR1 of the amino acid sequence described in SEQ ID NO: 32, an HC CDR2 of the amino acid sequence described in SEQ ID NO: 33, an HC CDR3 of the amino acid sequence described in SEQ ID NO: 34, an LC CDR1 of the amino acid sequence described in SEQ ID NO: 37, an LC CDR2 of the amino acid sequence described in SEQ ID NO: 38, and an LC CDR3 of the amino acid sequence described in SEQ ID NO: 39, or a substitution of 1, 2, 3, 4, 5, or 6 amino acids in the CDR region. In one non-limiting embodiment, the anti-TMPRSS6 antibody disclosed herein is the antibody identified herein as a humanized anti-TMPRSS6 antibody variant hzMWTx-001Var, comprising an HC polypeptide having the amino acid sequence described in SEQ ID NO: 73 and an LC polypeptide having the amino acid sequence described in SEQ ID NO: 75.

[0019] In one embodiment, the anti-TMPRSS6 antibody disclosed herein comprises a heavy chain (HC) variable region polypeptide having the amino acid sequence described in SEQ ID NO: 41, or a sequence substantially identical to that of SEQ ID NO: 41, and a light chain (LC) variable region polypeptide having the amino acid sequence described in SEQ ID NO: 46, or a sequence substantially identical to that of SEQ ID NO: 46. In one embodiment, the anti-TMPRSS6 antibody disclosed herein comprises a variant of the antibody having HC CDR1 of the amino acid sequence described in SEQ ID NO: 42, HC CDR2 of the amino acid sequence described in SEQ ID NO: 43, HC CDR3 of the amino acid sequence described in SEQ ID NO: 44, LC CDR1 of the amino acid sequence described in SEQ ID NO: 47, LC CDR2 of the amino acid sequence described in SEQ ID NO: 48, and LC CDR3 of the amino acid sequence described in SEQ ID NO: 49, or a substitution of 1, 2, 3, 4, 5, or 6 amino acids in the CDR region. In one non-limiting embodiment, the anti-TMPRSS6 antibody disclosed herein is the antibody identified herein as a humanized anti-TMPRSS6 antibody variant hzMWTx-002Var, comprising an HC polypeptide having the amino acid sequence described in SEQ ID NO: 77 and an LC polypeptide having the amino acid sequence described in SEQ ID NO: 79.

[0020] In one embodiment, the anti-TMPRSS6 antibody disclosed in the present disclosure comprises a heavy chain (HC) variable region polypeptide having the amino acid sequence set forth in SEQ ID NO: 51 or a sequence substantially identical to SEQ ID NO: 51, and a light chain (LC) variable region polypeptide having the amino acid sequence set forth in SEQ ID NO: 56 or a sequence substantially identical to SEQ ID NO: 56. In one embodiment, the anti-TMPRSS6 antibody disclosed in the present disclosure comprises HC CDR1 having the amino acid sequence set forth in SEQ ID NO: 52, HC CDR2 having the amino acid sequence set forth in SEQ ID NO: 53, HC CDR3 having the amino acid sequence set forth in SEQ ID NO: 54, LC CDR1 having the amino acid sequence set forth in SEQ ID NO: 57, LC CDR2 having the amino acid sequence set forth in SEQ ID NO: 58, and LC CDR3 having the amino acid sequence set forth in SEQ ID NO: 59, or variants of the above antibodies comprising substitutions of 1, 2, 3, 4, 5 or 6 amino acids in the CDR regions. In one non-limiting embodiment, the anti-TMPRSS6 antibody disclosed in the present disclosure is an antibody identified in the present disclosure as a humanized anti-TMPRSS6 antibody variant 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.

[0021] In another aspect, provided is an anti-TMPRSS6 antibody (including variants and fragments as disclosed in the present disclosure) that can be used to treat iron overload disorders, particularly disorders of iron metabolism such as β-thalassemia and other disorders of ineffective hematopoiesis. Methods and compositions for using the anti-TMPRSS6 antibody disclosed in the present disclosure are provided for therapeutic uses including, but not limited to, treating disorders of iron metabolism such as iron overload disorders, particularly β-thalassemia and other disorders of ineffective hematopoiesis. In certain embodiments, provided is a pharmaceutical composition comprising the anti-TMPRSS6 antibody disclosed in the present disclosure and a suitable carrier and / or excipient.

[0022] In another embodiment, a method for treating an iron metabolic disorder is provided, comprising administering an effective amount of the anti-TMPRSS6 antibody disclosed herein to a subject in need thereof, wherein the administration of an effective amount of the anti-TMPRSS6 antibody modulates the activity of components involved in iron metabolism. In a particular embodiment, a method for treating iron overload comprises administering an effective amount of the anti-TMPRSS6 antibody disclosed herein, wherein the administration of an effective amount of the anti-TMPRSS6 antibody modulates the activity of components involved in iron metabolism. In a particular embodiment, a method for treating iron overload comprises administering an effective amount of the anti-TMPRSS6 antibody disclosed herein, wherein the administration of an effective amount of the anti-TMPRSS6 antibody modulates the activity of at least one component involved in the regulation of hepcidin expression. In a particular embodiment, the method comprises inhibiting the suppression of hepcidin expression by TMPRSS6 by administering an effective amount of the anti-TMPRSS6 antibody. In a particular embodiment, the administration of an effective amount of the anti-TMPRSS6 antibody increases hepcidin expression. In certain embodiments, the method involves increasing the activity of the hepcidin promoter by administering an effective dose of anti-TMPRSS6 antibody. In certain embodiments, the method involves inhibiting the suppression of BMP / SMAD pathway-induced hepcidin expression by TMPRSS6 by administering an effective dose of anti-TMPRSS6 antibody. In certain embodiments, the method involves administering an effective dose of anti-TMPRSS6 antibody to a target that produces one or more biological effects associated with iron overload, including, but not limited to, decreased serum iron, decreased hepatic non-heme iron, increased serum hepcidin, increased hepatic hepcidin RNA, decreased splenomegaly, increased red blood cell count (RBC), increased hematocrit (HCT), decreased erythrocyte distribution width (RDW), and / or increased production of mature red blood cells (increased erythropoiesis).

[0023] In another embodiment, a method is provided for treating a disease or disease state involving abnormal suppression of hepcidin expression, the method comprising administering an effective dose of the anti-TMPRSS6 antibody disclosed herein to a subject in need thereof, the administration of an effective dose of the anti-TMPRSS6 antibody comprising modulating the activity of at least one component involved in the abnormal suppression of hepcidin expression, thereby mitigating the abnormal suppression of hepcidin expression. In certain embodiments, the method results in an increase in hepcidin expression.

[0024] In another embodiment, a method for treating an iron metabolic disorder related to suppressed hepcidin concentration is provided, the method comprising administering an effective amount of the anti-TMPRSS6 antibody disclosed herein to a subject in need thereof, wherein the administration of an effective amount of the anti-TMPRSS6 antibody modulates the activity of components involved in suppressing hepcidin concentration. In a particular embodiment, the method comprises administering an effective amount of the anti-TMPRSS6 antibody that increases serum hepcidin concentration, increases hepatic hepcidin RNA, and decreases serum iron concentration.

[0025] In another embodiment, a method is provided for treating a disorder of iron metabolism, including a disorder related to and / or characterized by ineffective hematopoiesis, which may include, but is not limited to, β-thalassemia. In this embodiment, the method comprises administering an effective dose of the anti-TMPRSS6 antibody disclosed herein to a subject known or suspected of having a disorder of iron metabolism related to and / or characterized by ineffective hematopoiesis, the administration resulting in one or more changes related to iron metabolism and / or erythropoiesis in the subject. In a particular embodiment, a method is provided in which the administration of an effective dose of the anti-TMPRSS6 antibody treats or improves at least one biological effect or symptom related to the disorder. In a particular embodiment, practicing the method results in one or more changes, including, but is not limited to, a decrease in hepatic non-heme iron, an increase in serum hepcidin, an increase in hepatic hepcidin RNA, a decrease in splenomegaly, an increase in red blood cell count (RBC), an increase in hematocrit (HCT), a decrease in erythrocyte distribution width (RDW), or an increase in the production of mature erythrocytes (increased erythropoiesis).

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

[0027] In another embodiment, one or more isolated nucleic acid molecules encoding at least one portion of the anti-TMPRSS6 antibody disclosed herein are provided. In certain embodiments, the isolated nucleic acid molecules encoding at least one portion of the anti-TMPRSS6 antibody disclosed herein include a nucleotide sequence listed in Table 1, Table 2, or Table 3, or a sequence substantially identical (e.g., at least 85%, 90%, 92%, 95%, 97%, or 98%, 99% identical) to a nucleotide sequence listed in Table 1, Table 2, or Table 3. In certain embodiments, an isolated nucleic acid molecule encoding at least one of the heavy chain (HC) sequences of the anti-TMPRSS6 antibody disclosed herein may include a nucleotide sequence selected from at least one of the following: Sequence ID No. 5 or substantially identical to Sequence ID No. 5, Sequence ID No. 15 or substantially identical to Sequence ID No. 15, Sequence ID No. 25 or substantially identical to Sequence ID No. 25, Sequence ID No. 35 or substantially identical to Sequence ID No. 35, Sequence ID No. 45 or substantially identical to Sequence ID No. 45, Sequence ID No. 55 or substantially identical to Sequence ID No. 55, Sequence ID No. 62 or substantially identical to Sequence ID No. 62, Sequence ID No. 66 or substantially identical to Sequence ID No. 66, Sequence ID No. 70 or substantially identical to Sequence ID No. 70, Sequence ID No. 74 or substantially identical to Sequence ID No. 74, Sequence ID No. 78 or substantially identical to Sequence ID No. 78, or Sequence ID No. 82 or substantially identical to Sequence ID No. 82.In certain embodiments, an isolated nucleic acid molecule encoding at least one light chain (LC) sequence of the anti-TMPRSS6 antibody or its antigen-binding fragment disclosed herein may include a nucleotide sequence selected from at least one of the following: SEQ ID NO: 10 or substantially identical to SEQ ID NO: 10, SEQ ID NO: 20 or substantially identical to SEQ ID NO: 20, or substantially identical to SEQ ID NO: 30 or substantially identical to SEQ ID NO: 30, SEQ ID NO: 40 or substantially identical to SEQ ID NO: 40, SEQ ID NO: 50 or substantially identical to SEQ ID NO: 50, SEQ ID NO: 60 or substantially identical to SEQ ID NO: 60, SEQ ID NO: 64 or substantially identical to SEQ ID NO: 64, SEQ ID NO: 68 or substantially identical to SEQ ID NO: 68; SEQ ID NO: 72 or substantially identical to SEQ ID NO: 72; SEQ ID NO: 76 or substantially identical to SEQ ID NO: 76; SEQ ID NO: 80 or substantially identical to SEQ ID NO: 80, or SEQ ID NO: 84 or substantially identical to SEQ ID NO: 84.

[0028] In another embodiment, a vector is provided comprising one or more nucleic acid molecules encoding at least one amino acid sequence of the anti-TMPRSS6 antibody disclosed herein. In a particular embodiment, a vector is provided comprising one or more nucleic acid molecules encoding at least one heavy chain (HC) or light chain (LC) sequence of the anti-TMPRSS6 antibody disclosed herein. In a particular embodiment, a vector is provided comprising a nucleic acid molecule encoding at least one portion of at least one of the amino acid sequences listed in Table 1, Table 2, or Table 3, or at least one portion of an amino acid sequence substantially identical to the amino acid sequences listed in Table 1, Table 2, or Table 3. In a particular embodiment, a vector is provided comprising a nucleic acid molecule encoding at least one portion of the HC or LC sequences listed in Table 1, Table 2, or Table 3, or at least one portion of an amino acid sequence substantially identical to the HC or LC sequences listed in Table 1, Table 2, or Table 3.

[0029] In another embodiment, at least one host cell is provided that contains a vector comprising one or more nucleic acid molecules encoding the amino acid sequence of the anti-TMPRSS6 antibody disclosed herein. In a particular embodiment, a host cell is provided that contains a vector comprising a nucleic acid molecule encoding at least one portion of at least one of the HC or LC sequences listed in Table 1, Table 2, or Table 3, or at least one portion of an amino acid sequence substantially identical to at least one of the HC or LC sequences listed in Table 1, Table 2, or Table 3. In a particular embodiment, at least one host cell is capable of supporting vector expression and recombinant production of the anti-TMPRSS6 antibody or its antigen-binding fragment encoded by the vector. In a particular embodiment, at least one host cell is capable of supporting vector expression and recombinant production of the anti-TMPRSS6 antibody or its antigen-binding fragment encoded by a vector comprising a nucleic acid molecule encoding at least one portion of at least one of the HC or LC sequences listed in Table 1, Table 2, or Table 3, or at least one portion of an amino acid sequence substantially identical to at least one of the HC or LC sequences listed in Table 1, Table 2, or Table 3. In certain embodiments, host cells are transiently transfected with a vector comprising one or more nucleic acid molecules encoding the amino acid sequence of an anti-TMPRSS6 antibody or its antigen-binding fragment disclosed herein, and the host cells are able to support vector expression and recombinant production of the anti-TMPRSS6 antibody or its antigen-binding fragment encoded by the vector. [Brief explanation of the drawing]

[0030] [Figure 1] This is the result of a cascade screening of anti-TMPRSS6 antibodies. Antibodies that bind to human TMPRSS6 were evaluated using an in vitro functional assay of HAMP promoter activity, and cross-reactivity with non-human TMPRSS6 was evaluated for antibodies that showed an effect on HAMP promoter activity. [Figure 2A-2F]Figures 2A-2F show the effect of anti-TMPRSS6 antibodies on HAMP promoter activity, as measured by a dual luciferase reporter assay performed on HepG2 cells, across a range of antibody concentrations. In each plot, white circles indicate results using anti-TMPRSS6 antibodies, and white squares indicate results using the same concentration of mouse IgG or human IgG1 as a negative (non-specific binding) control. Figure 2A shows the effect of MWTx-001 anti-TMPRSS6 antibody on HAMP promoter activity across a range of antibody concentrations. Figure 2B shows the effect of MWTx-002 anti-TMPRSS6 antibody on HAMP promoter activity across a range of antibody concentrations. Figure 2C shows the effect of MWTx-003 anti-TMPRSS6 antibody on HAMP promoter activity across a range of antibody concentrations. Figure 2D shows the effect of hzMWTx-001Var anti-TMPRSS6 antibody on HAMP promoter activity across a range of antibody concentrations. Figure 2E shows the effect of the hzMWTx-002Var anti-TMPRSS6 antibody on HAMP promoter activity across a range of antibody concentrations. Figure 2F shows the effect of the hzMWTx-003Var anti-TMPRSS6 antibody on HAMP promoter activity across a range of antibody concentrations. [Figure 3A-3M]Figures 3A-3M show the results of measuring the binding affinity of anti-TMPRSS6 antibodies. Figures 3A-3F show the results of measuring the binding affinity of anti-TMPRSS6 antibodies to human TMPRSS6 expressed on HEK293T cells using two different methods. In each plot, white circles represent the results of using anti-TMPRSS6 antibodies within a given concentration range, and white squares represent the results of using mouse IgG at the same concentration as a negative control. Figures 3A-3C show the results of measuring the binding of MWTx-001 (Figure 3A), MWTx-002 (Figure 3B), and MWTx-003 (Figure 3C) to human TMPRSS6 using cell surface ELISA (measurement of HRP-labeled secondary antibodies), with the calculated EC50 value of each antibody used as an estimate of the binding affinity. Figures 3D-3F show the results of measuring the binding of MWTx-001 (Figure 3D), MWTx-002 (Figure 3E), and MWTx-003 (Figure 3F) to human TMPRSS6 using FACS (measurement of APC-conjugated secondary antibodies), with the calculated EC50 value of each antibody used as an estimate of binding affinity. Figures 3G-3M show the results of measuring the affinity and binding kinetics of anti-TMPRSS6 antibodies to human ecto-TMPRSS6-FLAG using Octet® RED96e under analyte concentrations of 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.13 nM, 1.56 nM, and 0.78 nM. Figure 3G shows the binding kinetics of MWTx-001 anti-TMPRSS6 antibody to ecto-TMPRSS6-FLAG. Figure 3H shows the binding kinetics of the MWTx-002 anti-TMPRSS6 antibody to ecto-TMPRSS6-FLAG. Figure 3I shows the binding kinetics of the MWTx-003 anti-TMPRSS6 antibody to ecto-TMPRSS6-FLAG. Figure 3J shows the binding kinetics of the hzMWTx-001Var anti-TMPRSS6 antibody to ecto-TMPRSS6-FLAG. Figure 3K shows the binding kinetics of the hzMWTx-002Var anti-TMPRSS6 antibody to ecto-TMPRSS6-FLAG. Figure 3L shows the binding kinetics of the hzMWTx-003Var anti-TMPRSS6 antibody to ecto-TMPRSS6-FLAG. Figure 3M summarizes the affinity measurements for all anti-TMPRSS6 antibodies. [Figure 4A-4U]Figures 4A-4U show the results of measuring the cross-reactivity of anti-TMPRSS6 antibodies. Figures 4A-4I show the results of measuring the cross-reactivity of anti-TMPRSS6 antibodies MWTx-001, MWTx-002, and MWTx-003 to human TMPRSS6 and non-human TMPRSS6 expressed on HEK293T cells. Each histogram plot shows the results of FACS incubation of HEK293T cells expressing the TMPRSS6 target (thin line, lightly filled; shown with antibody name) with a single antibody, and the results of FACS incubation of control HEK293T cells that do not express the TMPRSS6 protein (thick line, darkly filled; shown with Ctrl) with the same antibody. Figures 4A to 4C show the results obtained when HEK293T cells stably expressing human TMPRSS6 (HuTMPRSS6-(His)6) were used together with MWTx-001 (Figure 4A), MWTx-002 (Figure 4B), and MWTx-003 (Figure 4C). Figures 4D to 4F show the results obtained when HEK293T cells stably expressing mouse TMPRSS6 (MoTMPRSS6-(His)6) were used together with MWTx-001 (Figure 4D), MWTx-002 (Figure 4E), and MWTx-003 (Figure 4F). Figures 4G to 4I show the results obtained when HEK293T cells transiently expressing cynomolgus monkey TMPRSS6 (CynoTMPRSS6-(His)6) were used together with MWTx-001 (Figure 4G), MWTx-002 (Figure 4H), and MWTx-003 (Figure 4I).Figures 4J-4U show the cross-reactivity of anti-TMPRSS6 antibodies against non-human (mouse (Figures 4J, 4L, 4N, 4P, 4R, 4T) or cynomolgus monkey (Figures 4K, 4M, 4O, 4Q, 4S, 4U)) TMPRSS6 expressed on HEK293T cells, using cell surface ELISA (measuring HRP-labeled secondary antibodies). The results were obtained for MWTx-001 anti-TMPRSS6 antibody (Figures 4J-4K) and MWTx-00 This figure shows the results of measuring the binding of 2 anti-TMPRSS6 antibodies (Figure 4L~4M), MWTx-003 anti-TMPRSS6 antibody (Figure 4N~4O), hzMWTx-001Var anti-TMPRSS6 antibody (Figure 4P~4Q), hzMWTx-002Var anti-TMPRSS6 antibody (Figure 4R~4S), and hzMWTx-003Var anti-TMPRSS6 antibody (Figure 4T~4U) to non-human TMPRSS6. In each plot, white circles indicate results using anti-TMPRSS6 antibodies, and white squares indicate results using mouse IgG or human IgG1 as negative (non-specific binding) controls. The EC50 value of each antibody is used as an estimate of binding affinity. [Figure 5A-5R] Figures 5A-5R show the results of FACS analysis of the binding of the anti-TMPRSS6 monoclonal antibodies MWTx-001 (Figures 5A-5C), MWTx-002 (Figures 5D-5F), MWTx-003 (Figures 5G-5I), and their humanized variants hzMWTx-001Var (Figures 5J-5L), hzMWTx-002Var (Figures 5M-5O), and hzMWTx-003Var (Figures 5P-5R) to HEK293T cells expressing homologous matriptase. HEK293T cells stably expressing human TMPRSS6 (matryptase-2) (Figures 5A, 5D, 5G, 5J, 5M, 5P) were used as positive controls. HEK293T cells overexpressing matryptase (ST14) (Figures 5B, 5E, 5H, 5K, 5N, 5Q) and / or matryptase-3 (TMPRSS7) (Figures 5C, 5F, 5I, 5L, 5O, 5R) proteins were used to test their binding to homologous matryptases. In each panel (Figures 5A-5R), HEK293T cells not expressing matryptase (HEK293T) were used as negative controls, clearly demonstrating the control (Ctrl) results. [Figure 6A-6L]Figures 6A-6L show that hepcidin expression in mice increases in a dose-dependent manner upon treatment with anti-TMPRSS6 antibody. Figures 6A-6C show the effects of MWTx-003 anti-TMPRSS6 antibody (Figures 6A-6B) or its humanized variant hzMWTx-003Var anti-TMPRSS6 antibody (Figure 6C) on serum iron. Figure 6D shows the effect of GFP-TMPRSS6 on serum hepcidin. Figures 6D-6F show the effects of MWTx-003 anti-TMPRSS6 antibody (Figures 6D-6E) or its humanized variant hzMWTx-003Var anti-TMPRSS6 antibody (Figure 6F) on serum hepcidin. Figure 6G shows the effect of GFP-TMPRSS6 on hepatic hepcidin RNA. Figures 6G-6I show the effects of MWTx-003 anti-TMPRSS6 antibody (Figures 6G-6H) or its humanized variant hzMWTx-003Var anti-TMPRSS6 antibody (Figure 6I) on hepatic hepcidin RNA. Figures 6J-6L show the serum concentrations of MWTx-003 anti-TMPRSS6 antibody (Figures 6J-6K) or its humanized variant hzMWTx-003Var anti-TMPRSS6 antibody (Figure 6L). Mouse IgG2b (MoIG2b) (Figures 6A-6B, 6D-6E, 6G-6H, 6J-6K) or human IgG1 (HuIGg1) (Figures 6C, 6F, 6I, 6L) were used as isotype controls, PBS was used as a vehicle control, and GFP vectors were used as vector controls (Figures 6A, 6D, 6G, 6J). [Figures 7A-7R]Figures 7A-7R show the in vivo efficacy of anti-TMPRSS6 antibodies using a β-thalassemia mouse model. Figures 7A-7D show the effects of MWTx-003 anti-TMPRSS6 antibodies on RBC (Figure 7A), HGB (Figure 7B), HCT (Figure 7C), and RDW (Figure 7D) using Th3 / + mice. Figure 7E shows the effect of MWTx-003 anti-TMPRSS6 antibodies on spleen weight using Th3 / + mice. Figure 7F shows the effect of MWTx-003 anti-TMPRSS6 antibodies on serum iron using Th3 / + mice. Figure 7G shows the effect of MWTx-003 anti-TMPRSS6 antibodies on liver non-heme iron using Th3 / + mice. Figure 7H shows the effect of MWTx-003 anti-TMPRSS6 antibodies on serum hepcidin using Th3 / + mice. Figure 7I shows the effect of MWTx-003 anti-TMPRSS6 antibody on hepatic hepcidin RNA using Th3 / + mice. Figure 7J shows the serum concentration of MWTx-003 anti-TMPRSS6 antibody using Th3 / + mice. Figures 7L-7M show the effect of MWTx-003 anti-TMPRSS6 antibody on erythrocyte production using bone marrow from Th3 / + mice. Figures 7O-7P show the effect of MWTx-003 anti-TMPRSS6 antibody on erythrocyte production using spleen cells from Th3 / + mice. The representative plots in Figures 7K-7P highlight four different cell clusters (I: basophilic erythroblasts, II: polychromic erythroblasts, III: normochromic erythroblasts and anucleated reticulocytes, IV: mature erythrocytes) and their corresponding proportions in cell numbers. Wild-type mice were used as positive controls (Figures 7A-7J, 7K, 7N), and mouse IgG2b (MoIgG2b) was used as an isotype control in the treatment (Figures 7A-7J, 7L, 7O). The bar graphs in Figures 7Q-7R show the mean results for cell clusters I, II, III, and IV in the bone marrow (Figure 7Q) and spleen (Figure 7R) for each treatment plan (WT, Th3 / +w / MoIgG2b, Th3 / +w / MWTx-003) after 4 weeks. Comparison allows for the identification of shifts in each population, particularly the shift to mature erythrocytes after MWTx-003 treatment. [Figures 8A-8D]Figures 8A-8D show the results of epitope binning of MWTx-001, MWTx-002, and MWTx-003 anti-TMPRSS6 antibodies against human ecto-TMPRSS6-FLAG using Octet® RED96e. Figure 8A shows the epitope binning of the MWTx-001 anti-TMPRSS6 antibody against ecto-TMPRSS6-FLAG. Figure 8B shows the epitope binning of the MWTx-002 anti-TMPRSS6 antibody against ecto-TMPRSS6-FLAG. Figure 8C shows the epitope binning of the MWTx-003 anti-TMPRSS6 antibody against ecto-TMPRSS6-FLAG. Figure 8D summarizes the association signals of the MWTx-001, MWTx-002, and MWTx-003 anti-TMPRSS6 antibodies. [Modes for carrying out the invention]

[0031] The present invention relates to a novel antibody that binds to TMPRSS6 and its antigen-binding fragment, as well as a method for producing and using the antibody that binds to TMPRSS6. Terms / Definitions

[0032] Scientific and technical terms used in connection with the present invention shall have meanings generally understood by those skilled in the art unless otherwise defined. The use of singular terms ("a," "an," "the," or other singular terms) shall include plural references, and plural terms shall include the singular form, unless the context indicates otherwise. For example, a reference to "antibody" includes "one or more" antibodies or "plural" such antibodies. All publications described herein are incorporated herein by reference in their entirety.

[0033] In general, the antibodies, antigen-binding fragments, compositions, and methods disclosed herein may employ nomenclature and techniques available to those skilled in the art in molecular biology, microbiology, cell and tissue culture, protein and nucleotide chemistry, and recombinant DNA technology. The techniques and procedures described herein are generally carried out in accordance with conventional methods well known in the art, as described in various general and more specific references, particularly Sambrook et al. (1989) MOLECULAR CLONING: A LABORATORY MANUAL (2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) and Ausubel et al. (1994) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Volumes I-III (John Wiley & Sons, NY). Enzymatic reactions and purification techniques are carried out in accordance with the manufacturer's specifications or methods generally achieved in the art, or the methods described herein, unless otherwise specifically stated herein. The preparation and formulation of pharmaceuticals, as well as the techniques and methods relating to the treatment of the subjects, are described in this disclosure using conventional nomenclature.

[0034] In a broad sense, an "antibody" refers to a polypeptide or combination of polypeptides that recognizes and binds to an antigen through one or more immunoglobulin variable regions. Here, the immunoglobulin variable regions can be naturally occurring or unnatural as a result of, for example, engineering, chimerization, humanization, optimization, CDR grafting, or affinity maturation.

[0035] The “antibodies” disclosed herein are whole (intact, full-length) antibodies, single-chain antibodies, or antigen-binding fragments having one or two chains, and may be of natural or non-natural origin. The antibodies have at least sufficient complementarity-determining regions (CDRs) interspersed with framework regions (FRs) and are capable of recognizing and binding to antigens. The anti-TMPRSS6 antibodies disclosed herein may be, but are not limited to, at least one of the following: monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, single-chain antibodies, Fab fragments, single-chain variable fragments (scFv), aptamers, single-domain antibodies (VHH or nano-substances), recombinant antibodies, modified antibodies with peptides / other sites added to the antibody, and / or modified antibodies with amino acids added to the N-terminus or C-terminus, or other TMPRSS6-binding fragments or variants. The terms whole antibody, full-length antibody, intact antibody, naturally occurring antibody, or equivalent terms are understood to refer to polypeptides, particularly glycoproteins, that contain at least two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds. Each HC consists of a heavy chain variable region (VH) and an HC constant region (CH), and each light chain consists of a light chain variable region (VL) and an LC constant region (CL). The variable regions of the HC and LC, VH and VL, contain binding domains that interact with antigens. The VH and VL regions can be further divided into a CDR region, which is characterized by hypervariability, and a generally more conserved FR region. Each VH and VL typically consists of three CDRs and four FRs arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The constant region of an antibody can mediate the binding of immunoglobulins to various cells of the immune system and to host tissues and factors, including the classical complement system. Typically, an antibody contains at least heavy chain (HC)CDR1, CDR2, CDR3 and light chain (LC)CDR1, CDR2, CDR3 sequences, one of which may be naturally occurring or non-natural. The antibody may have fewer CDR sequences as long as it can recognize and bind to the antigen.

[0036] The anti-TMPRSS6 antibodies disclosed herein may be variants comprising at least one altered CDR or framework sequence, the CDR and / or framework sequence may be optimized by mutating the nucleic acid molecule encoding such framework sequence. It is also possible to construct variants in which the HC and LC portions originate from independently different sources. Techniques for constructing variants include, but are not limited to, conservative amino acid substitution, computer modeling, screening of candidate polypeptides individually or in combination, and codon optimization, and those skilled in the art will understand that antibody variants can be constructed as needed. The anti-TMPRSS6 antibodies disclosed herein may also be fragments. The antigen-binding function of the antibody may be a monovalent fragment consisting of a Fab fragment, VL, VH, CL, and CH1 domain, F(ab )2 The antigen-binding portion can be fulfilled by segments such as fragments, bivalent fragments containing two Fab fragments disulfide-linked at the hinge region, Fd fragments consisting of VH and CH1 domains, single-strand variable fragments (scFv) consisting of the VL and VH domains of one arm of the antibody, single-domain antibody (dAb) fragments consisting of the VH domain, isolated CDRs (VHH, nanobodies), or aptamers. The antigen-binding portion can be incorporated into single-domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136). The antigen-binding portion of an antibody can be grafted onto a polypeptide-based scaffold to form a monobody (see, e.g., U.S. Patent No. 6,703,199 describing a fibronectin polypeptide monobody).

[0037] The term antibody encompasses a broad range of biochemically distinguishable classes of polypeptides. The “class” of an antibody refers to the type of constant domain or region in its heavy chain. Those skilled in the art will understand that antibodies have five major classes: IgA, IgD, IgE, IgG, and IgM, some of which are further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, each well-characterized and known to confer functional specificity. Modified versions of these classes and isotypes are readily identifiable and within the scope of this disclosure. While all immunoglobulin classes are within the scope of this disclosure, this disclosure will be primarily directed toward the IgG class of immunoglobulin molecules.

[0038] A chimeric antibody is an antibody in which a portion of the heavy chain (HC) and / or light chain (LC) involved in the formation of the immune reaction site originates from a specific source or species, while the remainder of the HC and / or LC originates from a different source or species. In certain embodiments, the target-binding region or site is of non-human origin (e.g., mouse or non-human primate), while the constant region is human.

[0039] As used herein, the term “humanized antibody” refers to an antibody or antibody variant derived from a non-human antibody, typically a mouse monoclonal antibody, where the parent non-human antibody-derived CDR is grafted (fused) onto a human immunoglobulin framework, particularly a framework containing a variable region derived from the Acceptor Human Framework or Human Consensus Framework. Techniques and principles for the design, production, and testing of humanized antibodies are known (Jones PT, Dear PH, Foote J, Neuberger MS, Winter G. Replacing complementarity-determining regions in human antibody with those from a mouse. Nature. 1986 May 29-Jun 4;321(6069):522-5; Almagro JC, Fransson J. Humanization of Antibodies. Front Biosci. 2008 Jan 1;13:1619-33). It is understood that modifications to the acceptor framework can be made at multiple locations to develop humanized antibodies with improved characteristics depending on the desired application, such as high affinity for the target, low clearance, and low toxicity. The anti-TMPRSS6 antibody disclosed herein may be a humanized variant.

[0040] "Affinity" refers to the sum of the strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, the binding affinity used in this disclosure refers to endogenous binding affinity, which reflects the 1:1 interaction between members of a binding pair (e.g., antibody and antigen). Affinity can be measured by common methods known in the art, including those described in this disclosure. Calculated concentration (calculated EC) at which approximately 50% of maximum binding occurs. 50 The affinity of molecule X to its partner Y can be estimated using the dissociation constant (Kd or KD), which is the k value measured for the interaction. off / k on It can be represented by (representing).

[0041] The “subjects” are mammals, which include, but are not limited to, primates (e.g., humans and non-human primates such as monkeys), livestock (e.g., cattle, sheep, cats, dogs, pigs, llamas, horses), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the subjects are humans. The phrases “to subjects who need it,” “to patients who need it,” “to patients who need treatment,” or “to subjects who need treatment” may include subjects who would benefit from the administration of the anti-TMPRSS6 antibody disclosed herein for the treatment of iron overload. It is understood that the administration of the anti-TMPRSS6 antibody to “subjects who need it” can be interpreted as referring to subjects who are known or suspected of having iron overload, particularly β-thalassemia, based on indicators such as symptoms, family history, and genotype. Furthermore, anti-TMPRSS6 antibodies may be administered to subjects who are not known or suspected of having impaired iron metabolism for prophylactic or preventive purposes, screening purposes, diagnostic purposes, research purposes, or to obtain results different from those obtained through treatment of the disorder, but are not limited to these purposes.

[0042] The “effective dose” of anti-TMPRSS6 antibody means, for example, in a pharmaceutical formulation, the amount effective in the dosage and duration required to obtain the desired therapeutic or prophylactic effect. It is understood that “effective dose” is intended to refer to the amount of anti-TMPRSS6 antibody or a pharmaceutical composition containing anti-TMPRSS6 antibody that elicits a biological response or desired therapeutic effect in the cells, tissues, systems, non-human animal subjects, non-human mammalian subjects, or human subjects being measured. The terms “therapeutic effective dose,” “pharmacological effective dose,” and “physiological effective dose” are used interchangeably to refer to the amount of anti-TMPRSS6 antibody required to provide a threshold level of the active ingredient in the bloodstream or within the target tissue. The exact amount depends on many factors, such as the specific anti-TMPRSS6 antibody (activator), the components and physical properties of the composition, the intended population of subjects / patients being treated, the subject’s medical condition, age, sex, and weight, etc., and a person skilled in the art would be able to readily determine it based on the information provided in this disclosure or other information available in relevant literature. In this context, the terms “improvement,” “increase,” or “decrease” refer to values ​​or parameters relative to baseline measurements, such as measurements in the same subject before the initiation of the treatment described in this disclosure, or measurements in a control individual (or multiple control individuals) without the treatment described in this disclosure.

[0043] The terms “pharmaceutical composition” or “pharmaceutical preparation” refer to a preparation in which the active ingredients contained herein, in particular anti-TMPRSS6 antibodies, are in a form that enables their biological activity. A pharmaceutical composition may include two or more active ingredients, for example, two or more anti-TMPRSS6 antibodies, or a combination of an anti-TMPRSS6 antibody and another active ingredient that acts on a different target. Such combinations may include, but are not limited to, an anti-TMPRSS6 antibody and another active ingredient that has a desired effect on hematopoietic processes, particularly erythrocyte production; an anti-TMPRSS6 antibody and a gene therapy agent, such as a gene therapy agent that targets the HBB gene; or an anti-TMPRSS6 antibody and an Fc fusion protein that targets TGF superfamily ligands and promotes erythrocyte production. A “pharmaceutically acceptable carrier” refers to a component in a pharmaceutical preparation other than the active ingredients that is non-toxic to the target. A pharmaceutically acceptable carrier may, but is not limited to, a buffer, excipient, stabilizer, adjuvant, or preservative.

[0044] As used herein, “treatment” or “to treat” or similar terms may refer to any outcome considered beneficial to a particular subject in a defined set of circumstances. Treating an iron metabolism disorder non-exclusively means reducing, improving, slowing, interrupting, preventing, mitigating, stopping, or reversing the progression or severity of an existing symptom, disorder, condition, or disease, and may also include preventing or delaying the onset of one or more symptoms of iron overload, and / or reducing the severity and frequency of one or more symptoms of iron overload. The terms “to treat” or “a method of treating” or equivalents may include, but are not limited to, one or more uses of the anti-TMPRSS6 antibody disclosed herein, including but not limited to therapeutic, prophylactic, diagnostic, imaging, and screening uses.

[0045] As used in this disclosure, the term "vector" refers to a nucleic acid molecule that can grow a nucleic acid, to which a vector sequence is linked, within a host cell into which the vector has been introduced. In this disclosure, a vector capable of inducing the expression of nucleic acids to which they are operably linked is referred to as an "expression vector." Anti-TMPRSS6 antibody

[0046] Antibodies and antigen-binding fragments are provided that bind to TMPRSS6 on the cell surface and can modulate the activity of at least one component involved in iron metabolism, particularly at least one component involved in iron overload associated with abnormal suppression of hepcidin expression. An anti-TMPRSS6 antibody that binds to TMPRSS6 on the cell surface and can modulate the activity of at least one component involved in the regulation of hepcidin expression can be used in methods for treating iron overload associated with abnormal suppression of hepcidin expression. An anti-TMPRSS6 antibody that binds to TMPRSS6 on the cell surface and can modulate the suppression of hepcidin expression by TMPRSS6 can be used to therapeutically target TMPRSS6 in methods for treating iron overload associated with abnormal suppression of hepcidin expression.

[0047] Once an antibody or fragment specific to TMPRSS6, particularly human TMPRSS6 expressed on the cell surface, is obtained, the desired biological activity of regulating the activity of at least one component involved in its iron metabolism can be tested by several methods known to those skilled in the art.

[0048] The terms “modulate” or “to modulate,” as used in this disclosure, or similar terms, are understood to refer to one or more effects that may occur when the anti-TMPRSS6 antibody disclosed herein binds to its target. “Modify” and its equivalent expressions may refer to different actions and effects depending on the component considered. That is, “modulate” means neutralizing, reversing, inhibiting, blocking, reducing, antagonizing, or otherwise interfering with the activity of a particular component involved in iron metabolism, while for other components involved in iron metabolism, the term modulate may mean increasing, enhancing, or agonizing these components.

[0049] It should be understood that the term "component" can refer not only to the target molecule TMPRSS6, but also to downstream processes and pathways involved in iron metabolism. Therefore, a component in the sense of a process or pathway may, but is not limited to, the regulation of hepcidin expression, TMPRSS6 inhibition of hepcidin expression, the process of hepcidin expression, the regulation of hepcidin concentration, the increase of hepcidin concentration, the activation of the hepcidin promoter, or the inhibition of BMP / SMAD pathway-induced hepcidin expression by TMPRSS6, the regulation of hepatic non-heme iron concentration, one or more processes involved in splenomegaly, or one or more hematopoietic processes involved in the regulation of red blood cell count (RBC), hematocrit (HCT), erythrocyte distribution width (RDW), and erythropoiesis, particularly the formation of mature red blood cells.

[0050] The anti-TMPRSS6 antibodies disclosed herein can be used to therapeutically target at least one component involved in iron metabolism, particularly at least one component involved in iron overload. In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein can be used to therapeutically target at least one component involved in the regulation of hepcidin expression and to modulate the activity of the component to achieve increased hepcidin expression. In certain embodiments, the anti-TMPRSS6 antibodies disclosed herein can be used to modulate the activity of the hepcidin promoter to achieve increased hepcidin expression. It is understood that the anti-TMPRSS6 antibodies disclosed herein can therapeutically target TMPRSS6 and thereby modulate the downstream activity of other components of hepcidin expression, such as, but not limited to, the regulation of hepatic non-heme iron concentration, one or more processes involved in splenomegaly, or one or more hematopoietic processes involved in the regulation of red blood cell count (RBC), hematocrit (HCT), erythrocyte distribution width (RDW), and erythropoiesis, particularly the production of mature red blood cells.

[0051] By using the anti-TMPRSS6 antibody disclosed herein, precise modulation of the target component is possible by therapeutically targeting at least one component involved in iron metabolism. It is understood that by using the anti-TMPRSS6 antibody disclosed herein to precisely target TMPRSS6 and its downstream effects on at least one component involved in the regulation of hepcidin expression, undesirable effects, delivery and / or efficacy difficulties, and regulatory hurdles associated with other approaches to the treatment of iron overload currently in use or under development, such as blood transfusions that may further exacerbate iron overload, iron chelation with poor patient compliance, intrusive phlebotomy or splenectomy that merely manage symptoms, gene therapies targeting HBB genes that may have permanent and multifaceted effects in multiple systems, gene therapies and gene editing with unknown off-target effects, Fc fusion proteins targeting TGF superfamily ligands to inhibit SMAD signaling that do not reduce the need for iron chelation therapy to manage iron overload, and other approaches that are difficult to control and deliver, such as hepcidin mimetic drugs, antisense or iRNA drugs targeting TMPRSS6. It is understood that using anti-TMPRSS6 antibodies for precise therapeutic targeting does not rule out the possibility of using anti-TMPRSS6 antibodies in combination therapies, such as combinations with other active ingredients acting on different targets, combinations with antibodies that bind to different targets, combinations with gene therapies and methods targeting HBB genes, or combinations with Fc fusion proteins targeting TGF superfamily ligands that stimulate erythrocyte production.

[0052] The anti-TMPRSS6 antibody disclosed herein can be used to develop targeted therapies (e.g., dosage, frequency of administration), therapies that are easy to continue and discontinue, and therapies that can be used in combination with other therapies. In one strategic embodiment, the anti-TMPRSS6 antibody disclosed herein can be combined with other therapies that can address multiple therapeutic targets and / or address the shortcomings or undesirable effects of any of the therapies in combination therapy. Exemplary embodiments of anti-TMPRSS6 antibody and its applications

[0053] Non-limiting exemplary embodiments of the anti-TMPRSS6 antibody of the present invention are shown herein, particularly disclosed in the examples, tables, and figures. Antibodies capable of binding to TMPRSS6

[0054] As shown in the examples, a functional cascade can be used to identify and characterize the anti-TMPRSS6 antibodies of the present invention. The first step of the cascade comprises screening antibodies that can bind to human TMPRSS6 on the surface of cells expressing TMPRSS6 (Example 1, Figure 1), and the second step comprises identifying antibodies that can bind to human TMPRSS6 on the surface of cells expressing TMPRSS6 and modulate the activity of components involved in iron metabolism, in which case they are tested for their ability to increase hepcidin (HAMP) promoter activity (Example 2). As shown by the exemplary embodiment shown in Figure 1, the first step identified 143 antibodies (clones) that can bind to human TMPRSS6 on the surface of cells expressing TMPRSS6, and the second step identified 10 antibodies (of the 143 screened) as “active” antibodies (clones) that were able to increase hepcidin (HAMP) promoter activity.

[0055] In the third stage of the functional cascade (Figure 1), ten “active” antibodies were tested for cross-reactivity with non-human TMPRSS6 targets from sources relevant to further research. Specifically, cross-reactivity with mouse TMPRSS6 relevant to preclinical efficacy studies in a mouse model was tested, and cross-reactivity with cynomolgus monkey TMPRSS6 relevant to toxicity (safety) studies was tested. As demonstrated by the exemplary embodiment shown in Figure 1, shown in Example 4, and shown in Figure 4, three (3) clones (of the ten screened) showed cross-reactivity with at least one non-human TMPRSS6 and were named MWTx-001, MWTx-002, and MWTx-003. The sequencing of each monoclonal antibody was determined, and the CDRs on each HC and LC were identified (Kabat numbering). The HC and LC sequences were identified as follows: MWTx-001 (SEQ ID NOs. 61(HC) and 63(LC)), MWTx-002 (SEQ ID NOs. 65(HC) and 67(LC)), and MWTx-0039 (SEQ ID NOs. 69(HC) and 71(LC)). Hybridoma cell lines producing the MWTx-001 monoclonal antibody were deposited on May 27, 2020, under the provisions of the Budapest Convention, with the American Type Culture Collection (ATCC®), 10801 University Boulevard, Manassas, Virginia, 20110, USA, under ATCC Accession No. PTA-126759. The hybridoma cell line producing the MWTx-002 monoclonal antibody was deposited on May 27, 2020, under the provisions of the Budapest Convention, with the American Type Culture Collection (ATCC®), 10801 University Boulevard, Manassas, Virginia, 20110, USA, under ATCC Accession No. PTA-126760.The hybridoma cell line producing the MWTx-003 monoclonal antibody was deposited on May 27, 2020, under the provisions of the Budapest Convention, with the American Type Culture Collection (ATCC®), 10801 University Boulevard, Manassas, Virginia, 20110, USA, under ATCC Accession No. PTA-126761. Humanized variant

[0056] Humanized antibodies containing a human-derived antibody framework grafted with a non-human-derived CDR are expected to be non-immunogenic when administered to humans. As demonstrated by the exemplary embodiments disclosed in Example 2, humanized anti-TMPRSS6 antibody variants were successfully generated, tested, optimized, and selected. For each HC or LC variant, multiple HC and LC variant candidates were developed in which the CDR sequence was the same, but the variable region framework sequence differed at more than 90% of the framework positions. These variants were tested in different HC / LC combinations to identify combinations with desirable characteristics. Following initial design and testing, some parental CDR sequences were modified to avoid potential undesirable events such as aspartate isomerization, and some constant regions (Fc) were modified to achieve desired functions, such as minimizing antibody-dependent cell-mediated cytotoxicity (ADCC). Variants showing the desired antigen-binding affinity were selected for further evaluation and development, resulting in the humanized variants hzMWTx-001Var (SEQ ID NOs. 73(HC) and 75(LC)), hzMWTx-002Var (SEQ ID NOs. 77(HC) and 79(LC)), and hzMWTx-003Var (SEQ ID NOs. 81(HC) and 83(LC)). Anti-TMPRSS6 antibody that increases hepcidin promoter activity

[0057] As disclosed herein, antibodies for use in the treatment of iron overload characterized by reduced hepcidin expression can modulate the activity of at least one component involved in hepcidin expression, which may be the activity of the hepcidin promoter. As demonstrated by exemplary embodiments using the in vitro assay disclosed in Example 2, the anti-TMPRSS6 antibodies MWTx-001, MWTx-002, MWTx-003, hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var increased HAMP promoter activity in a dose-dependent manner (Figures 2A-2F), while isotype controls at the same concentrations did not increase HAMP promoter activity. Anti-TMPRSS6 antibodies that exhibit high affinity for the target in the relevant biological context.

[0058] The anti-TMPRSS6 antibody showed high affinity for a biologically appropriate target, namely human TMPRSS6 expressed on the cell surface. As illustrated by exemplary embodiments of affinity measurements using three different methods disclosed in Example 3 and Figure 3M, the monoclonal antibodies MWTx-001, MWTx-002, and MWTx-003, as well as the humanized variants hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var, consistently exhibit suitable affinity properties for therapeutically effective antibodies or antibody fragments. Anti-TMPRSS6 antibody exhibiting cross-reactivity with non-human targets

[0059] For therapeutically useful antibodies or antibody fragments, it is desirable that the antibody or antibody fragment has sufficient cross-reactivity with non-human targets (non-human homologs) from sources that would be relevant to further research such as preclinical efficacy studies, disease animal models, and toxicity studies, so that it recognizes, for example, mouse homologs and / or primate homologs such as those derived from cynomolgus monkeys. As demonstrated by the exemplary embodiments disclosed in Example 4, MWTx-001, hzMWTx-001Var, MWTx-003, and hzMWTx-003Var showed detectable cross-reactivity with mouse TMPRSS6, while MWTx-001, MWTx-002, MWTx-003, hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var showed detectable cross-reactivity with cynomolgus monkey TMPRSS6. The anti-TMPRSS6 antibody specifically binds to TMPRSS6 (matryptase-2).

[0060] Antibodies that exhibit high specificity to target proteins and low cross-reactivity with homologous proteins within the same organism are expected to have little to no off-target effects. The anti-TMPRSS6 antibodies provided herein exhibit high specificity for human TMPRSS6 (matryptase-2), making them suitable for use in target compositions and methods. As demonstrated by the exemplary embodiments disclosed in Example 5 and shown in Figures 5A-R, the monoclonal antibodies MWTx-001, MWTx-002, and MWTx-003, and their humanized variants hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var, specifically bound to human TMPRSS6 (matryptase-2) and showed no cross-reactivity with the same type of human matryptase. In other words, these antibodies did not show detectable binding to matryptase-1 (ST14) or matryptase-3 (TMPRSS7). Anti-TMPRSS6 antibody with in vivo dose-dependent effects on hormones and symptoms associated with iron overload

[0061] Antibodies that can increase the concentration of serum hepcidin, a hormone that controls iron absorption and transfer from stored iron, are expected to alleviate, improve, or prevent the symptoms of iron overload, particularly the symptoms of elevated serum iron levels. As demonstrated by the exemplary embodiments shown in Example 6, administration of the anti-TMPRSS6 monoclonal antibody MWTx-003 or the humanized variant hzMWTx-003Var to wild-type subjects, i.e., subjects not known or suspected of having iron overload, resulted in increased serum hepcidin concentration (Figures 6A-6C), decreased serum iron concentration (Figures 6D-6F), and increased hepatic hepcidin RNA concentration (Figures 6G-6I) compared to isotype controls. These effects were dose-dependent, which can be interpreted as indicating that, regardless of the mechanism of action, the dose-dependent in vivo effect of the anti-TMPRSS6 antibody allows those skilled in the art to determine an effective dose for a given subject. Anti-TMPRSS6 antibody effective in vivo in a β-thalassemia disease model

[0062] Antibodies and antibody fragments that can alleviate the symptoms of iron overload in vivo when administered to subjects exhibiting animal models of iron overload, i.e., subjects known or suspected of having iron overload, are expected to have clinical therapeutic effects. As demonstrated by the exemplary embodiment shown in Example 7 using a β-thalassemia Th3 / + mouse model, administration of the anti-TMPRSS6 monoclonal antibody MWTx-003 compared to an isotype control resulted in multiple effects, including a decrease in hepatic non-heme iron, an increase in serum hepcidin, an increase in hepatic hepcidin RNA, suppression of splenomegaly, an increase in red blood cell count (RBC), an increase in hematocrit (HCT), a decrease in red blood cell distribution width (RDW), and an increase in mature red blood cell production (increased erythropoiesis). Each of these effects can be understood as an improvement in the symptoms of the disorder. The symptoms of this disease are expressed in multiple biological systems, including, but are not limited to, the liver (effects on hepatic non-heme iron and hepatic hepcidin RNA), the blood (effects on serum iron concentration, circulating hormone concentrations, especially serum hepcidin concentration, RBCs, HCT, and RDW), spleen size and function (splenomegaly), and, but are not limited to, erythropoiesis in multiple sites including the bone marrow and spleen (effects on the abundance of different progenitor cell types and mature erythrocytes in erythropoiesis sites). Administration of anti-TMPRSS6 antibody improved multiple symptoms in the disease model subjects, and the measured symptom levels shifted from levels seen in isotype controls (untreated disease) of the disease model to levels seen in wild-type littermates showing normal levels in genetically similar subjects that are not known or suspected of having the disease. Regardless of theory or mechanism of action, it is understood that ineffective hematopoiesis is the driving force behind increased iron absorption and abnormal suppression of hepcidin, which leads to iron overload, and that therapies that improve erythroblast differentiation and maturation into erythrocytes are therapeutically beneficial for the treatment of iron overload. This non-limiting exemplary embodiment discloses an anti-TMPRSS6 antibody therapy that increases erythroblast differentiation and maturation into erythrocytes and further reduces iron load. composition

[0063] The present invention provides compositions comprising a safe and effective amount of the anti-TMPRSS6 antibody and a pharmaceutically acceptable carrier or excipient(s) suitable for the intended use of each composition. Such carriers include, but are not limited to, physiological saline, buffer, glucose, water, glycerol, ethanol, excipients, stabilizers, preservatives, or combinations thereof. It is understood that pharmaceutical formulations should be tailored to the dosage form.

[0064] The anti-TMPRSS6 antibodies disclosed herein can be administered by any suitable means, including but not limited to injection or parenteral infusion. Parenteral infusion may include intramuscular, intravenous, intra-arterial, intraperitoneal, subcutaneous, or hepatic parenteral administration. The anti-TMPRSS6 antibodies disclosed herein can be formulated for local delivery to target tissue, or for introduction into liver tissue or the vascular system. The anti-TMPRSS6 antibodies disclosed herein can be administered using a device, as a depot, or in a sustained-release formulation (e.g., a semipermeable matrix of a solid hydrophobic polymer containing the antibody, or in microcapsules), and can be delivered slowly, and / or measured, and / or locally. The anti-TMPRSS6 antibodies disclosed herein can be formulated and administered using colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions. method

[0065] Methods for treating iron metabolism disorders using an effective dose of the anti-TMPRSS6 antibody disclosed herein are provided. Methods for targeting TMPRSS6 using the anti-TMPRSS6 antibody disclosed herein, without being constrained by a specific mechanism of action, result in multiple downstream effects, particularly on components (molecules, systems, processes) involved in iron metabolism and erythropoiesis. Methods for treating iron metabolism disorders using an effective dose of the anti-TMPRSS6 antibody disclosed herein are provided to modulate the activity of components involved in iron metabolism, without being constrained by a specific mechanism of action. In particular, methods are provided for treating iron overload disorders associated with excessive iron accumulation in tissues and organs, including, but not limited to, β-thalassemia, non-transfusion-dependent thalassemia, MDS (myelodysplastic syndrome), pure red blood cell anemia, and sideroblastic anemia, and disorders associated with or characterized by ineffective hematopoiesis. A method is provided for treating iron overload accompanied by low hepcidin levels, particularly diseases involving suppression of hepcidin expression (including diseases or conditions involving abnormal suppression of hepcidin expression), by administering an anti-TMPRSS6 antibody that can increase hepcidin expression without being limited to a single mechanism of action.

[0066] A method for treating iron metabolic disorders presented in this disclosure comprises administering an effective dose of the anti-TMPRSS6 antibody disclosed herein to a subject in need thereof, wherein the administration of an effective dose of the anti-TMPRSS6 antibody improves at least one of the biological effects (symptoms) involved in iron metabolism. A method is provided for treating iron metabolic disorders associated with suppressed hepcidin concentration, wherein administration of an effective dose of the anti-TMPRSS6 antibody disclosed herein to a subject in need thereof results in at least one of increased hepcidin promoter activity, increased hepcidin transcription, increased hepcidin RNA concentration, and increased hepcidin concentration (particularly serum hepcidin concentration). A method is provided for treating subjects known or suspected of having iron overload, wherein by administering an effective dose of anti-TMPRSS6 antibody, one or more biological effects are produced, but are not limited to, a decrease in hepatic non-heme iron, an increase in serum hepcidin, an increase in hepatic hepcidin RNA, a decrease in splenomegaly, an increase in red blood cell count (RBC), an increase in hematocrit (HCT), a decrease in erythrocyte distribution width (RDW), and an increase in the production of mature red blood cells (increased erythropoiesis). A method is provided for treating subjects known or suspected of having iron overload, characterized by ineffective hematopoiesis, wherein by administering an effective dose of anti-TMPRSS6 antibody, one or more biological effects are produced, but are not limited to, a decrease in hepatic non-heme iron, an increase in serum hepcidin, an increase in hepatic hepcidin RNA, a decrease in splenomegaly, an increase in red blood cell count (RBC), an increase in hematocrit (HCT), a decrease in erythrocyte distribution width (RDW), and an increase in the production of mature red blood cells (increased erythropoiesis).

[0067] Methods and compositions are provided for treating iron metabolism disorders, particularly iron overload disorders, and more particularly iron overload disorders characterized by ineffective hematopoiesis, wherein administration of an effective dose of anti-TMPRSS6 antibody results in the treatment or improvement of one or more biological effects or symptoms associated with the disorder. Regardless of theory or mechanism of action, it is understood that ineffective hematopoiesis, characterized by a low number of mature erythrocytes produced in the bone marrow by apoptosis of erythrocyte precursors, is the driving force behind increased iron absorption and abnormal suppression of hepcidin, which causes iron overload. According to this understanding, treatments that improve erythroblast differentiation and maturation into erythrocytes are considered beneficial for the treatment of iron overload. The efficacy of anti-TMPRSS6 antibody therapy in increasing erythroblast differentiation and maturation into erythrocytes, decreasing iron load, and increasing hepcidin expression maximizes the therapeutic benefits of the methods and compositions using the anti-TMPRSS6 antibody disclosed herein.

[0068] The following examples are provided to illustrate the invention of the claims and are not intended to limit the invention of the claims. [Examples]

[0069] Example 1: Antibody preparation and identification of antibodies that bind to TMPRSS6 Novel monoclonal antibodies against TMPRSS6 were produced under contract from the LakePharma Discovery Immunology Group (LakePharma, Inc., San Carlos, CA) using in vivo rodent immunization and hybridoma technology. DNA-based immunization was performed in B6;SJL mice (The Jackson Laboratories) using a mixture of pLEV113_huTMPRSS6 and pLEV113_moTMPRSS6-TCE plasmid DNA (cloned at LakePharma, Inc.) via hydrodynamic tail vein injection. Sufficient plasma titers were obtained by fluorescence-activated cell sorting (FACS), allowing for the commencement of downstream antibody recovery and screening activities. Splenocytes pooled from two immunized mice and myeloma fusion partners were fused using a NEPA GENE ECFG21 Super Electro Cell Fusion Generator (NEPAGINE Co., Ltd., Ichikawa City, Chiba Prefecture). The fusion material was plated into a total of 10 384-well plates of hypoxanthine-aminopterin-thymidine medium, which specifically selects hybridomas from unfused myeloma partner cells. Hybridoma supernatants were first screened for HuTMPRSS6 reactivity by FACS measurement. Supernatants that showed a positive staining signal on TMPRSS6-expressing HEK293T cells (selected by transfecting HEK293T cells with a plasmid encoding huTMPRSS6-(His)6 (SEQ ID NO: 97)) and negative staining on the parent nucleus (HEK293T) 10 days post-fusion were detected. Hybridoma supernatants that showed a positive staining signal on TMPRSS6-expressing HEK293 cells and a negative staining signal on the parent cells were considered "hits" and further screening was performed. 192 hits were identified in the primary FACS screening, and 143 hits were identified in the secondary and tertiary FACS screenings.

[0070] Example 2: Functional screening of anti-TMPRSS6 antibodies, identification, preparation, and sequencing of monoclonal anti-TMPRSS6 antibodies and humanized variants. HAMP-luciferase reporter assay

[0071] The response of the HAMP promoter to various anti-TMPRSS6 antibodies was measured using a hepcidin promoter-luciferase reporter assay (Du, X. et al., 2008. Science 320:1088-1092; modified to use a human HAMP promoter instead of a mouse HAMP promoter as originally disclosed). For the HAMP-luciferase reporting assay, a 2.5 kb HAMP promoter fragment (Reference Genome GRCh38) was spliced ​​upstream of the sequence encoding firefly luciferase. A control construct encoding Renilla luciferase driven by a thymidine kinase promoter (Promega, E6931) was used as an internal control. These constructs, along with the construct encoding TMPRSS6, were co-introduced into HepG2 cells (ATCC, HB-8065). Transfected HepG2 cells expressing TMPRSS6 were pretreated for approximately 3 hours with various concentrations of purified mAb diluted in starvation medium containing minimal essential medium (MEM, ATCC) + 1% thermo-inactivated fetal bovine serum (FBS, Gibco) + 1 mM sodium pyruvate + non-essential amino acid solution (Gibco) + 10 mM HEPES (Gibco) + 1% Pen / Strep (Gibco). They were then treated with recombinant hBMP6 (R&D Systems) at a final concentration of 25–60 ng / ml to induce BMP-SMAD-mediated signaling. Purified mouse IgG (Sigma-Aldrich) or human IgG1 (BioXcell) were used as controls. After overnight treatment with hBMP6, cells were lysed and luciferase substrates were added. Luminescence measurements of firefly luciferase and renila luciferase were recorded by measuring total luminescence. Activity was calculated as the ratio of firefly luciferase luminescence to renila luciferase luminescence (control). The results of these assays are shown in Figures 2A-2F. In vitro functional screening

[0072] To screen for functionally active hybridomas, all 143 HuTMPRSS6-binding hybridomas ("hits") were tested using the HAMP-luciferase reporter assay described above. Ten of the 143 HuTMPRSS6-binding hybridomas showed increased HAMP promoter activity (data not shown) and were identified as "active clones" for further testing. These ten active clones were tested for cross-reactivity to the mouse target MoTMPRSS6, as described in Example 4 below. By FACS, three showed binding to both HuTMPRSS6 and MoTMPRSS6. These three cross-reactive clones were further plated into 192 wells of a 384-well plate at a density of 1 cell / well to generate monoclonal hybridoma clones. Of the resulting subclones, those that showed desirable functional activity and cross-reactivity towards non-human targets, such as mouse TMPRSS6 (moTMPRSS6) and cynomolgus monkey TMPRSS6 (cynoTMPRSS6), were identified as MWTx-001, MWTx-002, and MWTx-003. Sequences of anti-TMPRSS6 antibodies MWTx-001, MWTx-002, and MWTx-003

[0073] The sequences of MWTx-001, MWTx-002, and MWTx-003 were determined by isolating mRNA from each hybridoma sample, amplifying the target variable region using reverse transcription polymerase chain reaction (RT-PCR) with a proprietary mouse IgG-specific primer set, and then sequencing the results. For each anti-TMPRSS6 antibody, unique heavy and light chains were identified. The nucleotide sequences of each heavy and light chain were determined. The amino acid sequences encoded by the nucleotide sequences were determined, and the CDR region was identified using the Kabat numbering system. Table 1 shows the amino acid sequences of the heavy and light chain variable regions, as well as the amino acid sequences of the identified CDR (based on Kabat numbering), and the nucleotide sequences of the heavy and light chain variable regions for MWTx-001, MWTx-002, and MWTx-003, respectively. [Table 1-1] [Table 1-2] [Table 1-3] Humanized anti-TMPRSS6 antibody variant generation and screening

[0074] Humanization of the parent antibody was performed by grafting CDRs onto a human antibody framework. First, homology modeling of the parent antibody's three-dimensional structure was performed to establish a structural model of the parent antibody. The amino acid sequence of the variable fragment framework was identified based on overall sequence identity, consistency of the VH-VL interface position, canonical position of similarly classified CDRs, and removal of candidate N-glycosylation sites. Humanized antibodies were designed by creating multiple hybrid sequences by fusing selected portions of the parent antibody sequence with human framework sequences. The isotype selected to format the humanized antibodies was IgG1 for the heavy chain and IgG1 kappa for the light chain. Using a 3D model, these humanized sequences were methodologically analyzed visually and by computer modeling to isolate sequences that were likely to retain antigen binding. The goal of the final humanized antibody was to maximize the amount of human sequence while maintaining the specificity of the original antibody. Subsequently, humanized variants paired with humanized VH and VL were expressed and purified for affinity analysis.

[0075] In one round of variant design, generation, and testing as part of affinity analysis, four VH variants were created by placing the VH-CDR of the parent antibody MWTX-003 at corresponding positions on four different human IgG1-derived frameworks (SEQ ID NOs: 89-92), and four VL(VK) variants were created by placing the VL-CDR of the parent antibody MWTX-003 at corresponding positions on four different human IgG1 kappa-derived frameworks (SEQ ID NOs: 93-96). A total of 16 humanized variants representing all combinations of VH and VL(VK) variants were prepared according to a 4VHx4VK matrix, and their antigen-binding properties (k on , k off Upon evaluating the KD (Kitting Discharge), it was found that the KD value was 4.16E-07 (~1.09E-08), which falls within the nanomolar range.

[0076] Variants exhibiting desirable antigen-binding affinity were selected and further evaluated and developed. In some cases, the parental CDR sequence was modified to avoid potential undesirable events such as aspartate isomerization.

[0077] To suppress the effector function of antibodies, particularly antibody-dependent cytotoxicity (ADCC), key amino acid residues in the Fc region were identified and mutated (substituted) in all humanized antibody variants. Guidelines obtained from publications on Fc mutations to achieve the goal of ADCC elimination are described in this mutation, for example, (Tamm A, Schmidt RE. IgG binding sites on human Fc gamma receptors. Int Rev Immunol. 1997;16(1-2):57-85.doi:10.3109 / 08830189709045703; Jefferis R, Lund J. Interaction sites on human IgG-Fc for FcgammaR: current models. Immunol Lett. 2002 Jun 3;82(1-2):57-65.doi:10.1016 / s0165-2478(02)00019-6) regarding the native Fc in hIgG1. It is used to indicate mutations such as the removal of the N-linked glycosylation site (N297A mutation) or the substitution of leucine at positions 234 and 235 of the lower hinge region in Fc (LALA double mutation). In this modified example, the N297A mutation was introduced into the Fc of the hzMWTx-001Var antibody and the hzMWTx-002Var antibody, and the LALA mutation was introduced into the Fc of the hzMWTx-003Var antibody, achieving the same goal of reducing or suppressing ADCC (Table 3, SEQ ID NOs: 73, 77, 81).

[0078] Based on the evaluation, the humanized anti-TMPRSS6 antibody variants hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var were selected for further testing. The sequences and characteristics of the humanized variants are shown in Tables 2 and 3 below. Recombination of humanized anti-TMPRSS6 antibody variants

[0079] The expression construct for the humanized anti-TMPRSS6 antibody variant was designed by incorporating an internal ribosome entry site (IRES) between the LC-coding DNA sequence and the HC-coding DNA sequence. The codons were optimized using Geneart DNA synthesis, and the construct was cloned into a pcDNA3.4 mammalian expression vector (ThermoFisher). The sequence of the inserted DNA was confirmed by sequencing. For recombinant antibody production, the ExpiCHO expression system (ThermoFisher) was used, and the expression construct was used for transient transfection according to the manufacturer's instructions. The expressed antibody was purified by protein A affinity chromatography. The antibody yield from transient transfection ranged from 50 mg to 300 mg per liter, with a purity of over 95% and an endotoxin level of less than 1 EU / ml. Sequences of humanized anti-TMPRSS6 antibody variants hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var

[0080] The humanized anti-TMPRSS6 antibody variants hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var were selected for further testing. The sequences of each variable region are shown in Table 2 below. Here, the identified CDRs are underlined, and the changes made to the humanized variant CDR sequence compared to the parent antibody are shown and discussed. [Table 2-1] [Table 2-2] [Table 2-3]

[0081] Table 3 shows the complete heavy and light chain protein and nucleotide sequences of the anti-TMPRSS6 monoclonal antibodies MWTx-001, MWTx-002, and MWTx-003, as well as the humanized anti-TMPRSS6 antibody variants hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var. The heavy chain protein sequences of the humanized anti-TMPRSS6 antibody variants hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var indicate the location of the mutations (changes) introduced to reduce ADCC as described above. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] Dose-dependent effect of anti-TMPRSS6 antibody on HAMP promoter activity

[0082] Figures 2A - 2F show the results of using the above - described HAMP luciferase reporter assay to test MWTx - 001, MWTx - 002, MWTx - 003 and their humanized variants hzMWTx - 001Var, hzMWTx - 002Var, hzMWTx - 003Var at the indicated concentrations. MWTx - 001 (Figure 2A), MWTx - 002 (Figure 2B), MWTx - 003 (Figure 2C) and the humanized variants hzMWTx - 001Var (Figure 2D), hzMWTx - 002Var (Figure 2E), hzMWTx - 003Var (Figure 2F) increase HAMP promoter activity in a dose - dependent manner. The EC 50 of MWTx - 001 was calculated to be 3 μg / ml (Figure 2A). The EC 50 of MWTx - 002 was calculated to be 1 μg / ml (Figure 2B). The EC 50 of MWTx - 003 was calculated to be 2 μg / ml (Figure 2C). The EC 50 of hzMWTx - 001Var was calculated to be 0.8 μg / ml (Figure 2D). The EC 50 of hzMWTx - 002Var was calculated to be 0.3 μg / ml (Figure 2E). The EC 50 of hzMWTx - 003Var was calculated to be 0.3 μg / ml (Figure 2F).

[0083] Example 3: Binding Affinity of Anti - TMPRSS6 Antibodies The binding affinities of various anti - TMPRSS6 antibodies to human TMPRSS6 expressed on HEK293T cells were measured using three different methods: cell - surface ELISA (Figures 3A - 3C), FACS (Figures 3D - 3F), and biolayer interferometry (Figures 3G - 3M). Measurement of Binding Affinity of Anti - TMPRSS6 mAb Using Cell - Surface ELISA

[0084] HEK293T cells stably expressing human TMPRSS6 (produced by LakePharma Inc. as described above; SEQ ID NO: 97) were fixed with 4% paraformaldehyde (PFA), washed with dPBS (Dulbecco phosphate-buffered saline, Corning Cellgro), and incubated with various concentrations of anti-TMPRSS6 antibody diluted in BSA medium (DMEM + 1% Pen / Strep + 10mM HEPES + 1 mg / ml BSA (Sigma-Aldrich)). Purified mouse IgG was used as a control (Sigma-Aldrich). After incubation, cells were washed with BSA medium and subsequently incubated with goat anti-mouse IgG conjugated with HRP as a 2° antibody (Invitrogen). Finally, cells were washed with dPBS to remove unbound antibody, developed with ELISA liquid substrate (Sigma-Aldrich), and then stopped by adding an equal volume of 1 M H2SO4 ELISA liquid substrate. The bound antibody was OD 450nm The absorbance was measured. The results of these assays are shown in Figures 3A-3C. Measurement of anti-TMPRSS6 mAb binding affinity using FACS

[0085] HEK293T cells stably expressing human TMPRSS6 were harvested, blocked with dPBS + 3% BSA, and incubated with various concentrations of anti-TMPRSS6 antibody diluted with dPBS + 3% BSA. Purified mouse IgG was used as a control. After incubation, the cells were washed with dPBS and subsequently incubated with goat anti-mouse IgG conjugated with APC as a 2° antibody (Jackson ImmunoResearch Inc). Finally, the cells were washed with dPBS to remove unbound antibody, resuspended in dPBS + 1 mM EDTA, and then subjected to FACS analysis using a NOVOCYTE® Flow Cytometer (ACEA Biosciences, Inc., San Diego, CA). The conjugated antibody was determined by excitation at 640 nm, emission (fluorescence) at 675 nm, and measurement of the average APC intensity. The results of these assays are shown in Figures 3D-3F. Measurement of affinity and binding rate of anti-TMPRSS6 antibody using bio-layer interferometry.

[0086] Bio-Layer Interferometry technology using the Octet® RED96e system (Sartorius AG) was used to measure the affinity and binding rate of the anti-TMPRSS6 antibody. A pre-hydrated anti-mouse IgGFc capture (AMC) biosensor (for MWTx-001, MWTx-002, MWTx-003 anti-TMPRSS6 antibodies, Figures 3G-3I) or anti-human IgG Fc capture (AHC) biosensor (hzMWTx-001Var, hzMWTx-002Var, and hzMWTx-003Var anti-TMPRSS6 antibodies, Figures 3J-3L) was first mixed with 1xKB (kinetic buffer, 1xPBS). The analytes were equilibrated in pH 7.4 + 0.02% Tween-20 + 0.1% BSA) for 120 seconds to establish the initial baseline. Subsequently, 10 mg / ml anti-TMPRSS6 antibody (MWTx-001, Figure 3G; MWTx-002, Figure 3H; MWTx-003, Figure 3I; hzMWTx-001Var, Figure 3J; hzMWTx-002Var, Figure 3K; hzMWTx-003Var, Figure 3L) was loaded onto an AMC or AHC biosensor for 240 seconds. Next, after establishing a second baseline signal for 120 seconds, the analytes were associated with various concentrations of human ecto-TMPRSS6-FLAG (SEQ ID NO: 102) (in-house produced by fusing the extracellular domain of human TMPRSS6 with a FLAG tag at the C-terminus) for 240 seconds. Finally, the analytes were lysed in 1 x KB for 360 seconds. Data analysis was performed using Octet Data Analysis HT Software. KD, k on , k off and R 2 This is summarized in Figure 3M.

[0087] Example 4: Cross-reactivity: Binding of anti-TMPRSS6 antibody to human TMPRSS6 and non-human TMPRSS6. Cross-reactivity determination using FACS

[0088] We tested whether the selected anti-TMPRSS6 antibody could bind to TMPRSS6 derived from mouse and / or cynomolgus monkeys. We used HEK293T cells (generated by LakePharma Inc. as described above) that stably express human TMPRSS6 (HuTMPRSS6-(His)6), and mouse TMPRSS6 (MoTMPRSS6-(His)6). )6 HEK293T cells (SEQ ID NO: 98) (generated by LakePharma Inc. as described above) that stably express ) and cynomolgus monkey TMPRSS6 (CynoTMPRSS6-(His )6 HEK293T cells transiently expressing (SEQ ID NO: 99) (in-house developed) were collected. HEK293T cells stably expressing human TMPRSS6 were used as a positive control, and HEK293T cells were used as a negative control (as described above). The cells were blocked with dPBS + 3% BSA and then incubated with anti-TMPRSS6 antibody diluted with dPBS + 3% BSA. After incubation, the cells were washed with dPBS and subsequently incubated again with goat anti-mouse IgG conjugated with AlexaFluor-488 as a 2° antibody (Invitrogen). Finally, the cells were washed with dPBS to remove unbound antibody, resuspended in dPBS + 1 mM EDTA, and then FACS analysis was performed using a NOVOCYTE® Flow Cytometer (ACEA Biosciences, Inc., San Diego, CA). The bound antibody was determined by excitation at 488 nm and measurement of emission (FITC-A) at 530 nm. The results of these assays are shown in the histogram plots in Figures 4A-4I. Cross-reactivity with mouse TMPRSS6 was observed in MWTx-001 (Figure 4D) and MWTx-003 (Figure 4F), but MWTx-002 (Figure 4E) did not show any detectable cross-reactivity with mouse TMPRSS6. Cross-reactivity with cynomolgus monkey TMPRSS6 was observed in MWTx-001 (Figure 4G), MWTx-002 (Figure 4H), and MWTx-003 (Figure 4I). Cross-reactivity determination using cell surface ELISA method

[0089] HEK293T cells stably expressing mouse TMPRSS6 (generated by LakePharma Inc. as described above; Figures 4J, 4L, 4N, 4P, 4R, 4T) or cynomolgus monkey (in-house prepared as described above; Figures 4K, 4M, 4O, 4Q, 4S, 4U) were fixed with methanol (100%), washed with dPBS (Dulbecco's phosphate-buffered saline, Corning Cellgro), and then incubated with various concentrations of anti-TMPRSS6 antibodies and their humanized variants diluted in BSA medium (DMEM + 1% Pen / Strep + 10mM HEPES + 1 mg / ml BSA (Sigma-Aldrich)). Purified mouse IgG (Figures 4J-4O) or human IgG1 (Figures 4P-4U) were used as controls. After incubation, the cells were washed in BSA medium and then incubated with goat anti-mouse (Invitrogen, Figures 4J-4O) or anti-human (Millipore, Figures 4P-4U) IgG conjugated with HRP as a 2° antibody. Finally, the cells were washed with dPBS to remove unbound antibodies, developed with ELISA liquid substrate (Sigma-Aldrich), and then stopped by adding an equal volume of 1M H2SO4 ELISA liquid substrate. The bound antibodies were OD 450nm The absorbance was measured. The results of these assays are shown in Figures 4J-4U. Cross-reactivity with mouse TMPRSS6 was observed with MWTx-001 (Figure 4J) and MWTx-003 (Figure 4N) anti-TMPRSS6 antibodies, as well as their humanized variants hzMWTx-001Var (Figure 4P) and hzMWTx-003Var (Figure 4T) anti-TMPRSS6 antibodies. On the other hand, MWTx-002 (Figure 4L) anti-TMPRSS6 antibody or its humanized variant hzMWTx-002Var (Figure 4R) anti-TMPRSS6 antibody did not show detectable cross-reactivity with mouse TMPRSS6. Cross-reactivity with cynomolgus monkey TMPRSS6 was observed with MWTx-001 (Figure 4K), MWTx-002 (Figure 4M), and MWTx-003 (Figure 4O) anti-TMPRSS6 antibodies, as well as their humanized variants hzMWTx-001Var (Figure 4Q), hzMWTx-002Var (Figure 4S), and hzMWTx-003Var (Figure 4U) anti-TMPRSS6 antibodies.

[0090] Example 5: Target specificity: Anti-TMPRSS6 antibody that binds to homologous matriptase. To determine whether the anti-TMPRSS6 antibody binds to homologous matriptases, HEK293T cells overexpressing matriptases (ST14) (SEQ ID NO: 100) (Figures 5B, 5E, 5H, 5K, 5N, 5Q) and HEK293T cells overexpressing matriptases-3 (TMPRSS7) (SEQ ID NO: 101) (Figures 5C, 5F, 5I, 5L, 5O, 5R) were collected (in-house produced). HEK293T cells stably expressing human TMPRSS6 (matriptases-2) (SEQ ID NO: 97) (generated by LakePharma Inc. as described above; Figures 5A, 5D, 5G, 5J, 5M, 5P) were used as positive controls, and HEK293T cells (Figures 5A-5R) were used as negative controls (as described above). Cells were blocked and permeabilized with dPBS + 3% BSA + 0.1% Tween-20 before incubation with various anti-TMPRSS6 antibodies diluted in dPBS + 3% BSA + 0.1% Tween-20. Cells were incubated with anti-TMPRSS6 antibodies and their humanized variants at a concentration of approximately 1 μg / ml for 1 hour. After incubation, cells were washed with dPBS and incubated with goat anti-mouse IgG conjugated with AlexaFluor-488 (Invitrogen, Figures 5A-5I) or goat anti-human IgG conjugated with allophycocyanin (APC), (Jackson Immuno Research, Figures 5J-5R) as the second antibody. Finally, cells were washed with dPBS, resuspended in dPBS + 1 mM EDTA, and then subjected to FACS analysis using a NOVOCYTE® Flow Cytometer. The bound antibodies were determined by excitation at 488 nm and measurement of emission at 530 nm (FITC-A) (Figures 5A-5I), or by excitation at 640 nm and measurement of emission at 675 nm (APC-A) (Figures 5J-5R). The results of these assays are shown in the histogram plots in Figures 5A-5R. All antibodies showed binding to human TMPRSS6 (matriptase-2) (Figures 5A, 5D, 5G, 5J, 5M, 5P), and none of the antibodies showed binding to homologous matriptase ST14 (Figures 5B, 5E, 5H, 5K, 5N, 5Q) or TMPRSS7 (Figures 5C, 5F, 5I, 5L, 5O, 5R).The MWTx-001 anti-TMPRSS6 antibody and its humanized variant, hzMWTx-001Var anti-TMPRSS6 antibody, showed binding to human TMPRSS6 (Figure 5A, 5J), but did not show binding to matryptase (ST14) (Figure 5B, 5K) or matryptase-3 (TMPRSS7) (Figure 5C, 5L). The MWTx-002 anti-TMPRSS6 antibody and its humanized variant, hzMWTx-002Var anti-TMPRSS6 antibody, showed binding to human TMPRSS6 (matryptase-2) (Figure 5D, 5M), but did not show binding to matryptase (ST14) (Figure 5E, 5N) or matryptase-3 (TMPRSS7) (Figure 5F, 5O). The MWTx-003 anti-TMPRSS6 antibody and its humanized variant, hzMWTx-003Var anti-TMPRSS6 antibody, showed binding to human TMPRSS6 (matryptase-2) (Figure 5G, 5P), but did not show binding to matryptase (ST14) (Figure 5H, 5Q) or matrytase-3 (TMPRSS7) (Figure 5I, 5R).

[0091] Example 6: Treatment with anti-TMPRSS6 antibody in a mouse pharmacodynamic model To study the in vivo pharmacodynamic response of anti-TMPRSS6 antibodies, 2–10 mg / kg of MWTx-003 anti-TMPRSS6 antibody (Figures 6A–6B, 6D–6E, 6G–6H, 6J–6K) or its humanized variant hzMWTx-003Var anti-TMPRSS6 antibody (Figures 6C, 6F, 6I, 6L) was intraperitoneally injected into wild-type C57BL / 6J mice. Mouse IgG2b (BioXcell, Figures 6A–6B, 6D–6E, 6G–6H, 6J–6K) or human IgG1 (BioXcell, Figures 6C, 6F, 6I, 6L) were used as isotype controls. Twenty hours after injection, 50 μg of GFP-TMPRSS6 plasmid DNA (prepared in-house by inserting human TMPRSS6 into a GFP vector) was delivered to 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 Biomiga's (San Diego, CA) EZgene Total RNA Purification Plus according to the manufacturer's instructions. Mouse serum was obtained by centrifugation of whole blood at 1500 × g for 10 minutes. The effect of anti-TMPRSS6 antibody treatment on serum iron levels.

[0092] Serum iron was measured using a colorimetric assay developed in-house (Figures 6A-6C). Briefly, mouse serum or standard iron (31-500 μg / dL) was mixed with a mixed acid solution (0.6 M trichloroacetic acid, 0.4 M sodium thioglycolate, 1 M HCl) by vertex for 30 seconds. The mixture was incubated at 37°C for 10 minutes, then centrifuged at 10,000 x g for 10 minutes, and colored with Color Solution (1.5 M sodium acetate, 0.5 mM bathophenanthroline disulfonate). Subsequently, OD (Oral Spectroscopy) was performed. 535nm Absorbance was measured. Serum iron concentration was calculated from a linear iron standard curve. Treatment with 10 mg / kg of MWTx-003 anti-TMPRSS6 antibody (Figures 6A-6B) and its humanized variant hzMWTx-003Var anti-TMPRSS6 antibody (Figure 6C) significantly reduced serum iron levels. The effect of anti-TMPRSS6 antibody treatment on serum hepcidin.

[0093] Serum hepcidin was measured using the Hepcidin-Murine Compete ELISA kit purchased from Intrinsic Lifesciences (La Jolla, CA) according to the manufacturer's instructions (Figures 6D-6F). Briefly, diluted mouse serum or hepcidin standards were mixed with hepcidin-biotin conjugates and then added to plates coated with anti-mouse hepcidin antibody. Serum hepcidin or hepcidin standards competed with the hepcidin-biotin conjugates and bound to the coated anti-hepcidin antibody. The bound hepcidin-biotin conjugates were detected with streptavidin-conjugated horseradish peroxidase (HRP), colored with TMB, and stopped with stop solution. Subsequently, OD (Oral-Density Specimen) was used. 450nm Absorbance was read. The data were analyzed using 4-parameter logistic (4-PL) curve fitting on Graphpad Prism 8, and serum hepcidin concentration was interpolated. Hydrodynamic delivery of GFP-TMPRSS6 significantly reduced serum hepcidin concentration (Figure 6D), but treatment with 10 mg / kg of MWTx-003 anti-TMPRSS6 antibody (Figures 6D-6E) and its humanized variant hzMWTx-003Var anti-TMPRSS6 antibody (Figure 6F) reversed the suppression of hepcidin and significantly increased serum hepcidin concentration. The effects of anti-TMPRSS6 antibody treatment on hepatic hepcidin RNA.

[0094] Hepatic hepcidin RNA was quantified by real-time qPCR (Figures 6G-6I). In short, cDNA was first synthesized from hepatic RNA using iScript Reverse Transcription Supermix (Bio-Rad) according to the manufacturer's instructions. The hepcidin transcript was amplified using the specific primers shown in Table 4 and detected using SsoAdvanced® Universal SYBR® Green Supermix (Bio-Rad) on a Bio-Rad CFX96 qPCR instrument according to the manufacturer's instructions. Samples were analyzed in triplicates, and the results were normalized to β-actin RNA levels (measured by transcription, amplification with the primers listed in Table 4, and quantification as described above). Hydrodynamic delivery of GFP-TMPRSS6 significantly reduced hepatic hepcidin RNA (Figure 6G). Treatment with 10 mg / kg of MWTx-003 anti-TMPRSS6 antibody (Figure 6G-6H) and its humanized variant hzMWTx-003Var anti-TMPRSS6 antibody (Figure 6I) reversed Hamp suppression and significantly increased hepatic hepcidin RNA concentration. The following primers were used for RNA quantification by real-time qPCR: Hepcidin forward primer: 5'-AAG CAG GGC AGA CAT TGC GAT-3' (SEQ ID NO: 85); Hepcidin reverse primer: 5'-CAG GAT GTG GCT CTA GGC TAT-3' (SEQ ID NO: 86); β-actin forward primer: 5'-ACC CAC ACT GTG CCC ATC TA-3' (SEQ ID NO: 87); β-actin reverse primer: 5'-CAC GCT CGG TCA GGA TCT TC-3' (SEQ ID NO: 88).

[0095] The serum concentrations of MWTx-003 anti-TMPRSS6 antibody or its humanized variant, hzMWTx-003Var anti-TMPRSS6 antibody, were quantified using a cell surface ELISA developed in-house (see Figures 6J-6L above). Briefly, diluted mouse serum or anti-TMPRSS6 antibody standards were incubated with 100% methanol-fixed HEK293T cells (HEK293T cells used as a background control) that stably express human TMPRSS6. The bound MWTx-003 anti-TMPRSS6 antibody was detected with HRP-bound goat anti-mouse IgG, and the bound hzMWTx-003Var anti-TMPRSS6 antibody was detected with HRP-bound goat anti-human IgG. After development with TMB, the mixture was stopped in stop solution. Subsequently, OD (Oral-Density Spectroscopy) was performed. 450nm Absorbance was read. Samples were analyzed in triplicate, and the results were standardized against the HEK293T control. Data were analyzed using 4-parameter logistic (4-PL) curve fitting in Graphpad Prism 8 to interpolate serum anti-TMPRSS6 antibody concentrations.

[0096] Example 7: In vivo efficacy of anti-TMPRSS6 antibody using a β-thalassemia mouse model To investigate the in vivo effects of the anti-TMPRSS6 antibody, we used a β-thalassemia mouse model (B6.129P2-Hbb-b1 tm1Unc Hbb-b2 tm1Unc We selected mice from JAX Stock No: 002683 (The Jackson Laboratories, Bar Harbor ME). These are referred to as Th3 / + mice in this book. 4-5 week old Th3 / + mice and their wild-type (WT) littermates were fed an iron-rich diet (Teklad TD.80394). Th3 / + mice were administered 10 mg / kg MWTx-003 anti-TMPRSS6 antibody or mouse IgG2b isotype control every 3 days for 4 weeks, while WT littermates were not. After the completion of the administration course, the mice were euthanized, and spleen, liver, femur, and blood samples were collected. Total RNA from the liver was purified, and serum was collected in the same manner as above.

[0097] Effects on blood cell count, splenomegaly, serum iron, serum hepcidin, and hepatic hepcidin RNA Complete blood count (CBC) was performed using a VETSCAN HM5 automated hematology counter (Figures 7A-7D). Treatment with the MWTx-003 anti-TMPRSS6 antibody significantly increased red blood cell count (RBC, Figure 7A) and hematocrit (HCT, Figure 7C) and reduced red blood cell distribution width (RDW, Figure 7D) in Th3 / + mice, but had no significant effect on hemoglobin (HGB, Figure 7B).

[0098] When spleen weight was measured, treatment with the MWTx-003 anti-TMPRSS6 antibody significantly suppressed splenomegaly in Th3 / + mice (Figure 7E).

[0099] Serum iron was measured in the same manner as described above. Treatment with MWTx-003 anti-TMPRSS6 antibody significantly reduced serum iron (Figure 7F). Non-heme iron in the liver was measured using a similar colorimetric assay (Figure 7G). Briefly, minced liver tissue was dried overnight at 65°C, then digested with mixed acid (3M HCl, 10% trichloroacetic acid) at 65°C for 20 hours. The supernatant was then collected and colored with Color Solution (1.5M thorium acetate, 0.5mM bathophenanthroline disulfonate). Subsequently, OD 535nm Absorbance was measured. Treatment with MWTx-003 anti-TMPRSS6 antibody significantly reduced non-heme iron in the liver (Figure 7G).

[0100] Serum hepcidin was measured using the hepcidin-murin competent ELISA kit, as described above. Treatment with MWTx-003 anti-TMPRSS6 antibody significantly increased serum hepcidin levels (Figure 7H).

[0101] Hepatic hepcidin RNA was quantified by real-time qPCR. Treatment with MWTx-003 anti-TMPRSS6 antibody significantly increased hepatic hepcidin RNA (Figure 7I).

[0102] The serum concentration of the MWTx-003 anti-TMPRSS6 antibody was quantified using a cell surface ELISA developed in-house, as described above (Figure 7J). Effects on red blood cell production

[0103] To investigate the effect of the MWTx-003 anti-TMPRSS6 antibody on erythropoiesis in Th3 / + mice, bone marrow was collected from the femur (see Figures 7K-7M), and spleen cells were collected from the spleen (see Figures 7N-7P) and analyzed. The collected cells were blocked with rat anti-mouse CD16 / CD32 (BD Biosciences) for 15 minutes, and then stained on ice for 30 minutes with FITC-conjugated rat anti-mouse TER119 (BD Biosciences) and APC-conjugated rat anti-mouse CD44 (Invitrogen). Washed cells were stained with the viability marker 7-AAD (BD Biosciences) on ice for 10 minutes before FACS analysis using a NOVOCYTE® Flow Cytometer. Ter119+ and 7-ADD- cells were selected, and density plots were graphed on cell size against anti-mouse CD44 (FSC-H). The plots were analyzed to identify cell types (cell clusters) and determine the abundance of each type (cluster). The representative plots in Figures 7K-7P show that four different cell clusters corresponding to the sequential stages of erythrocyte differentiation were distinguished from top to bottom and identified as follows: basophilic erythroblasts (Cluster I), polychromatic erythroblasts (Cluster II), orthochromatic erythroblasts and anucleated reticulocytes (Cluster III), and mature erythrocytes (Cluster IV). As shown in Figures 7K-7P, the percentage (%) value of each cluster in the sample was calculated as an indicator of the richness of cell types within that cluster. For each sample (bone marrow, spleen) of each animal in each treatment course, the percentage values ​​of each cell cluster (I), (II), (III), and (IV) were calculated as follows. The study included WT (untreated) mice (N=9), disease model Th3 / + mice treated with IgG2b isotype control (Th3+w / MoIgG2b), and disease model Th3 / + mice treated with MWTx-003 anti-TMPRSS6 antibody (N=5), with N=7 mice. The mean values ​​were calculated.After 4 weeks, on average, basophilic erythroblasts (I) shifted from 7.58% (Th3+w / MoIgG2b) to 6.52% (Th3+w / MWTx-003) (7.96% compared to WT), polychromatic erythroblasts (II) shifted from 54.20% (Th3+w / MoIgG2b) to 40.01% (Th3+w / MWTx-003) (28.53% compared to WT), orthochromatic erythroblasts and anucleated reticulocytes (III) shifted from 24.06% (Th3+w / MoIgG2b) to 29.73% (Th3+w / MWTx-003) (26.67% compared to WT), and mature erythrocytes (IV) shifted from 4.54% (Th3+w / MoIgG2b) to 16.44% (27.66% compared to WT). In the spleen, after 4 weeks, on average, basophilic erythroblasts (I) shifted from 0.71% (Th3+w / MoIgG2b) to 0.91% (Th3+w / MWTx-003) (0.46% compared to WT), and polychromatic erythroblasts (II) shifted from 45.76% (Th3+w / MoIgG2b) to 19.25% (Th3+w / MWTx-003) (12.23% compared to WT). Orthochromatic erythroblasts and anucleated reticulocytes (III) shifted from 31.16% (Th3+w / MoIgG2b) to 28.72% (Th3+w / MWTx-003) (8.67% compared to WT), and mature erythrocytes (IV) shifted from 14.13% (Th3+w / MoIgG2b) to 44.38% (Th3+w / MWTx-003) (72.17% compared to Wt). These results are shown as bar graphs in Figure 7Q for bone marrow and in Figure 7R for spleen.

[0104] In Th3 / + mice, treatment with the MWTx-003 anti-TMPRSS6 antibody improved ineffective hematopoiesis, and a significant percentage of erythroblasts differentiated and matured into erythrocytes.

[0105] Example 8: Anti-TMPRSS6 antibody epitope binning OCTET® RED96e was used for epitope binning of the MWTx-001 (Figure 8A), MWTx-002 (Figure 8B), and MWTx-003 (Figure 8C) anti-TMPRSS6 antibodies. First, ecto-TMPRSS6-FLAG (as described above) was labeled with biotin using the Biotinylation Kit (Abcam). A pre-hydrated streptavidin (SA) biosensor was equilibrated with 1x KB (as described above) for 60 seconds to establish the first baseline, and then 10 mg / ml of biotinylated ecto-TMPRSS6-FLAG was loaded onto the SA biosensor for 300 seconds. Next, a second baseline signal was established for 60 seconds before saturation with 50 mg / ml of antibody in 1x KB (MWTx-001, Figure 8A; MWTx-002, Figure 8B; MWTx-003, Figure 8C) for 600 seconds. Finally, a third baseline signal was established for 60 seconds before saturation with 50 μg / ml of MWTx-001, MWTx-002, or MWTx-003 in 1xKB for 300 seconds. Binding of the MWTx-001 anti-TMPRSS6 antibody to ecto-TMPRSS6-FLAG did not compete with the MWTx-002 anti-TMPRSS6 antibody or the MWTx-003 anti-TMPRSS6 antibody (Figure 8A). Binding of the MWTx-002 anti-TMPRSS6 antibody to ecto-TMPRSS6-FLAG did not compete with the MWTx-001 anti-TMPRSS6 antibody, but it did compete with the MWTx-003 anti-TMPRSS6 antibody (Figure 8B). The binding of the MWTx-003 anti-TMPRSS6 antibody to ecto-TMPRSS6-FLAG did not compete with the MWTx-001 anti-TMPRSS6 antibody, but it did compete with the MWTx-002 anti-TMPRSS6 antibody (Figure 8C). Data analysis was performed using Octet Data Analysis HT Software. Association signals are summarized in Figure 8D.

Claims

1. An anti-TMPRSS6 antibody that binds to human TMPRSS6 on the surface of cells expressing human TMPRSS6 and can modulate the activity of at least one component involved in iron metabolism.

2. The anti-TMPRSS6 antibody according to claim 1, wherein the anti-TMPRSS6 antibody can modulate the activity of at least one component involved in the regulation of hepcidin expression.

3. The anti-TMPRSS6 antibody according to claim 2, wherein the anti-TMPRSS6 antibody can substantially inhibit TMPRSS6 suppression of hepcidin expression, increase hepcidin expression, increase the activity of the hepcidin promoter, and substantially inhibit TMPRSS6 suppression of BMP / SMAD pathway-induced expression of hepcidin.

4. The anti-TMPRSS6 antibody according to claim 1, which exhibits cross-reactivity with at least one non-human TMPRSS6.

5. The anti-TMPRSS6 antibody according to claim 4, wherein the non-human TMPRSS6 is at least one of mouse TMPRSS6 or cynomolgus monkey TMPRSS6.

6. The anti-TMPRSS6 antibody according to claim 1, wherein the anti-TMPRSS6 antibody specifically binds to human TMPRSS6 (matryptase-2) and does not show detectable binding to a matryptase homolog selected from at least one of human matryptase-1 (ST14) or human matryptase-3 (TMPRSS7).

7. The anti-TMPRSS6 antibody according to claim 1, wherein the antibody is a monoclonal antibody.

8. The anti-TMPRSS6 antibody according to claim 1, wherein the antibody is a chimeric antibody.

9. The anti-TMPRSS6 antibody according to claim 1, wherein the antibody is a humanized antibody.

10. The anti-TMPRSS6 antibody according to claim 1, wherein the antibody is an antigen-binding fragment.

11. Sequence ID 1; Sequence ID 2; Sequence ID 3; Sequence ID 4; Sequence ID 6; Sequence ID 7; Sequence ID 8; Sequence ID 9; Sequence ID 11; Sequence ID 12; Sequence ID 13; Sequence ID 14; Sequence ID 16; Sequence ID 17; Sequence ID 18; Sequence ID 19; Sequence ID 21; Sequence ID 22; Sequence ID 23; Sequence ID 24; Sequence ID 26; Sequence ID 27; Sequence ID 28; Sequence ID 29; Sequence ID 31; Sequence ID 32; Sequence ID 33; Sequence ID 34; Sequence ID 36; Sequence ID 37; Sequence ID 38; Sequence ID 39; Sequence ID 41; Sequence ID 42; Sequence ID No. The anti-TMPRSS6 antibody according to claim 1, comprising at least one polypeptide having an amino acid sequence selected from No. 43; SEQ ID NO: 44; SEQ ID NO: 46; SEQ ID NO: 47; SEQ ID NO: 48; SEQ ID NO: 49; SEQ ID NO: 51; SEQ ID NO: 52; SEQ ID NO: 53; SEQ ID NO: 54; SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 58; SEQ ID NO: 59; SEQ ID NO: 61; SEQ ID NO: 63; SEQ ID NO: 65; SEQ ID NO: 67; SEQ ID NO: 69; SEQ ID NO: 71; SEQ ID NO: 73; SEQ ID NO: 75; SEQ ID NO: 77; SEQ ID NO: 79; SEQ ID NO: 81; or SEQ ID NO:

83.

12. The anti-TMPRSS6 antibody according to claim 1, wherein the antibody comprises (a) a heavy chain (HC) polypeptide in which the variable region comprises an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 1, a sequence substantially identical to SEQ ID NO: 1, the amino acids of SEQ ID NO: 31, and a sequence substantially identical to SEQ ID NO: 31, and (b) a light chain (LC) polypeptide in which the variable region comprises an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 6, a sequence substantially identical to the amino acids of SEQ ID NO: 6, the amino acids of SEQ ID NO: 36, and a sequence substantially identical to the amino acids of SEQ ID NO:

36.

13. The antibody according to claim 12, wherein the antibody comprises (a) a heavy chain including at least one of the heavy chain complementarity determination region 1 (HC CDR1) of SEQ ID NO: 2, HC CDR1 of SEQ ID NO: 32, heavy chain complementarity determination region 2 (HC CDR2) of SEQ ID NO: 33, heavy chain complementarity determination region 3 (HC CDR3) of SEQ ID NO: 4, and HC CDR3 of SEQ ID NO: 34; and (b) a light chain including at least one of the complementarity determination region 1 (LC CDR1) of SEQ ID NO: 7, LC CDR1 of SEQ ID NO: 37, light chain complementarity determination region 2 (LC CDR2) of SEQ ID NO: 8, LC CDR2 of SEQ ID NO: 38, light chain complementarity determination region 3 (LC CDR3) of SEQ ID NO: 9, and LC CDR3 of SEQ ID NO:

39.

14. The anti-TMPRSS6 antibody according to claim 1, wherein the antibody comprises (a) a heavy chain (HC) polypeptide in which the variable region comprises an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 11, a sequence substantially identical to SEQ ID NO: 11, the amino acids of SEQ ID NO: 41, and a sequence substantially identical to SEQ ID NO: 41, and (b) a light chain (LC) polypeptide comprising an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 16, a sequence substantially identical to SEQ ID NO: 16, the amino acid sequence of SEQ ID NO: 46, and a sequence substantially identical to the amino acid sequence of SEQ ID NO:

46.

15. The antibody according to claim 14, comprising: (a) a heavy chain including at least one of the heavy chain complementarity determination region 1 (HC CDR1) of SEQ ID NO: 12, HC CDR1 of SEQ ID NO: 42, heavy chain complementarity determination region 2 (HC CDR2) of SEQ ID NO: 13, HC CDR2 of SEQ ID NO: 43, heavy chain complementarity determination region 3 (HC CDR3) of SEQ ID NO: 14, and HC CDR3 of SEQ ID NO: 44; and (b) a light chain including at least one of the complementarity determination region 1 (LC CDR1) of SEQ ID NO: 17, LC CDR1 of SEQ ID NO: 47, light chain complementarity determination region 2 (LC CDR2) of SEQ ID NO: 18, LC CDR2 of SEQ ID NO: 48, light chain complementarity determination region 3 (LC CDR3) of SEQ ID NO: 19, and LC CDR3 of SEQ ID NO:

49.

16. The anti-TMPRSS6 antibody according to claim 1, wherein the antibody comprises (a) a heavy chain (HC) polypeptide in which the variable region comprises an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 21, a sequence substantially identical to SEQ ID NO: 21, the amino acids of SEQ ID NO: 51, and a sequence substantially identical to SEQ ID NO: 51; and (b) a light chain (LC) polypeptide comprising an amino acid sequence selected from the amino acid sequence of SEQ ID NO: 26, a sequence substantially identical to SEQ ID NO: 26, the amino acid sequence of SEQ ID NO: 56, and a sequence substantially identical to the amino acid sequence of SEQ ID NO:

56.

17. The anti-TMPRSS6 antibody according to claim 16, wherein the antibody comprises (a) a heavy chain including at least one of the heavy chain complementarity determination region 1 (HC CDR1) of SEQ ID NO: 22, HC CDR1 of SEQ ID NO: 52, heavy chain complementarity determination region 2 (HC CDR2) of SEQ ID NO: 23, HC CDR2 of SEQ ID NO: 53, heavy chain complementarity determination region 3 (HC CDR3) of SEQ ID NO: 24, and HC CDR3 of SEQ ID NO: 54; and (b) a light chain including at least one of the complementarity determination region 1 (LC CDR1) of SEQ ID NO: 27, LC CDR1 of SEQ ID NO: 57, light chain complementarity determination region 2 (LC CDR2) of SEQ ID NO: 28, LC CDR2 of SEQ ID NO: 58, light chain complementarity determination region 3 (LC CDR3) of SEQ ID NO: 29, and LC CDR3 of SEQ ID NO:

59.

18. A method for treating an iron metabolic disorder, comprising administering an effective amount of the anti-TMPRSS6 antibody according to claim 1 to a subject in need thereof, wherein the administration of an effective amount of the anti-TMPRSS6 antibody modulates the activity of components involved in iron metabolism.

19. The method according to claim 18, wherein the impaired iron metabolism is iron overload.

20. The method according to claim 19, wherein the aforementioned iron overload is impaired hematopoiesis.

21. The method according to claim 19, wherein the iron overload is β-thalassemia.

22. The method according to claim 18, wherein the activity of at least one component involved in the regulation of hepcidin expression is regulated by administration of an effective amount of anti-TMPRSS6 antibody.

23. The method according to claim 22, wherein the suppression of hepcidin expression by TMPRSS6 is inhibited by administering an effective amount of anti-TMPRSS6 antibody.

24. The method according to claim 22, wherein the expression of hepcidin is increased by administering an effective amount of anti-TMPRSS6 antibody.

25. The method according to claim 22, wherein the activity of the hepcidin promoter is increased by administering an effective amount of anti-TMPRSS6 antibody.

26. The method according to claim 22, wherein the suppression of BMP / SMAD pathway-induced hepcidin expression by TMPRSS6 is inhibited by administration of an effective amount of anti-TMPRSS6 antibody.

27. The method according to claim 18, wherein administration of an effective amount of anti-TMPRSS6 antibody results in at least one effect selected from: increased serum hepcidin concentration, decreased serum iron concentration, increased hepatic hepcidin RNA concentration, decreased hepatic non-heme iron, decreased splenomegaly, increased red blood cell count (RBC), increased hematocrit (HCT), decreased erythrocyte distribution width (RDW), increased mature erythropoiesis, and increased erythropoiesis.

28. A pharmaceutical composition comprising the anti-TMPRSS6 antibody described in claim 1 and a suitable carrier and / or excipient.

29. An isolated nucleic acid molecule encoding at least a portion of the anti-TMPRSS6 antibody described in claim 1.

30. An isolated nucleic acid molecule according to claim 29, comprising a heavy chain (HC) nucleotide sequence selected from a nucleotide sequence including sequence number 5, a nucleotide sequence including a sequence substantially identical to sequence number 5, a nucleotide sequence including sequence number 15, a nucleotide sequence including a sequence substantially identical to sequence number 15, a nucleotide sequence including sequence number 25, a nucleotide sequence including a sequence substantially identical to sequence number 25, a nucleotide sequence including sequence number 35, a nucleotide sequence forming a sequence substantially identical to sequence number 35, a nucleotide sequence including sequence number 45, a nucleotide sequence including a sequence substantially identical to sequence number 45, a nucleotide sequence including sequence number 55, or a nucleotide sequence including a sequence substantially identical to sequence number 55.

31. An isolated nucleic acid molecule according to claim 29, comprising a light chain (LC) nucleotide sequence selected from a nucleotide sequence including SEQ ID NO: 10, a nucleotide sequence substantially identical to SEQ ID NO: 10, a nucleotide sequence including SEQ ID NO: 20, a nucleotide sequence substantially identical to SEQ ID NO: 20, a nucleotide sequence including SEQ ID NO: 30, a nucleotide sequence substantially identical to SEQ ID NO: 30, a nucleotide sequence including SEQ ID NO: 40, a nucleotide sequence including SEQ ID NO: 40, a nucleotide sequence including SEQ ID NO: 560, or a nucleotide sequence substantially identical to SEQ ID NO:

60.

32. A vector comprising the nucleic acid molecule described in claim 29.

33. A host cell comprising the vector according to claim 32.

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