Anti-TMPRSS6 antibodies and uses thereof

Abstract

Provided herein are monoclonal antibodies that bind to the transmembrane serine protease 6 (TMPRSS6) protein, and methods of using the same. In various embodiments of the disclosure, the antibodies are fully human antibodies that bind to TMPRSS6. In some embodiments, the antibodies disclosed herein are useful in methods for treating or preventing diseases, disorders, or conditions associated with TMPRSS6 in humans.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 187,150, filed May 11, 2021, the entire disclosure of which is incorporated herein by reference.

[0002] Sequence Listing The contents of the Sequence Listing, submitted electronically as an ASCII text file (Filename: 728825_RGN9-002PC_ST25.txt; Size: 141.9 KB; Creation Date: May 10, 2022), are incorporated herein by reference in their entirety.

[0003] Technical Field Disclosed herein are antibodies and antigen-binding fragments thereof that specifically bind to transmembrane serine protease 6 (TMPRSS6). Also disclosed herein are nucleic acids comprising nucleotide sequences encoding such antibodies, host cells comprising same, and therapeutic methods using such antibodies and antigen-binding fragments thereof. [Background technology]

[0004] Transmembrane serine protease 6 (TMPRSS6; also known as matriptase-2 or MTP-2) is a type II transmembrane serine protease that consists of a short N-terminal intracellular region followed by a single transmembrane domain, a single SEA domain, two CUB domains, three LDLRa repeat domains, and a C-terminal catalytic serine protease domain. TMPRSS6 is highly expressed in the liver and plays a role in iron homeostasis by negatively regulating hepcidin expression. High levels of hepcidin reduce the levels of ferroportin, an iron efflux channel from the cell surface, blocking iron transport out of the cell. TMPRSS6 cleaves hemojuvelin (HJV), which results in decreased SMAD signaling and subsequent downregulation of transcription of HAMP, the gene that encodes hepcidin.

[0005] Attenuation of hepcidin expression may lead to iron overload or hemochromatosis. Iron overload disorder is a group of disorders that cause excess iron storage in the body. Since the body cannot excrete excess iron, excess iron is stored in certain organs, such as the liver, heart, and pancreas. Excess iron stored in such organs may cause organ damage and other related diseases, such as diabetes.

[0006] Examples of iron overload disorders include hereditary hemochromatosis (HHC), an inherited disease in which the body absorbs too much iron from the diet, and secondary hemochromatosis, which can occur as a complication in patients with certain blood disorders such as thalassemia (a type of inherited anemia caused by decreased hemoglobulin production) or myelodysplastic syndromes (blood cancers that result in acquired anemia). In the case of beta thalassemia and / or myelodysplastic syndromes, iron overload can often result from repeated blood transfusions to treat the anemia associated with the disease.

[0007] Treatment options for iron overload disorders are limited and typically require treatment for the patient's lifetime. Treatment options include iron chelation therapy, removal of iron-rich blood by phlebotomy or phlebotomy, and dietary restrictions to reduce iron intake. There is a need in the art for additional and improved compositions and methods for the treatment of iron overload disorders. Summary of the Invention

[0008] Disclosure Summary Disclosed herein are antibodies and antigen-binding fragments thereof that specifically bind to the transmembrane serine protease 6 (TMPRSS6) protein (i.e., anti-TMPRSS6 antibodies). In certain embodiments, the anti-TMPRSS6 antibodies are fully human antibodies that bind to human TMPRSS6 with high affinity and inhibit its protease activity.

[0009] In one aspect, the disclosure provides an isolated antibody or antigen-binding fragment thereof that specifically binds to transmembrane serine protease 6 (TMPRSS6) protein, wherein the antibody or antigen-binding fragment thereof binds to the catalytic domain of TMPRSS6 but does not bind to the catalytic triad of TMPRSS6.

[0010] In certain embodiments, the antibody or antigen-binding fragment thereof further binds at a site contained within LDLRa domain 2 of TMPRSS6. In one embodiment, the binding of the antibody or antigen-binding fragment thereof to TMPRSS6 does not directly occlude the catalytic triad of TMPRSS6. In other embodiments, the binding of the antibody or antigen-binding fragment thereof to TMPRSS6 mediates allosteric regulation of TMPRSS6. In certain embodiments, the binding of the antibody or antigen-binding fragment thereof to TMPRSS6 mediates allosteric inhibition of TMPRSS6.

[0011] In another aspect, the present disclosure provides an isolated antibody or antigen-binding fragment thereof that specifically binds to transmembrane serine protease 6 (TMPRSS6) protein, and the antibody or antigen-binding fragment thereof is an allosteric inhibitor of TMPRSS6. In certain embodiments, the antibody or antigen-binding fragment maintains TMPRSS6 in an inactive conformation that can bind to but not cleave a TMPRSS6 substrate. In certain embodiments, the TMPRSS6 substrate is hemojuvelin (HJV).

[0012] In another aspect, the disclosure provides an isolated antibody or antigen-binding fragment thereof that specifically binds to transmembrane serine protease 6 (TMPRSS6) protein, where the antibody or antigen-binding fragment binds within the catalytic domain of TMPRSS6 at a site that sterically occludes the catalytic triad of TMPRSS6. In certain embodiments, the antibody or antigen-binding fragment thereof competes with hemojuvelin (HHV) for binding to TMPRSS6.

[0013] In another aspect, the disclosure provides an isolated antibody or antigen-binding fragment thereof that specifically binds to human transmembrane serine protease 6 (hTMPRSS6) protein (SEQ ID NO: 126), wherein the antibody or antigen-binding fragment thereof interacts with one or more amino acids contained within the extracellular domain of hTMPRSS6 as determined by hydrogen / deuterium exchange.

[0014] In certain embodiments, the antibody or antigen-binding fragment thereof interacts with an amino acid sequence selected from (a) amino acids 125-133 of SEQ ID NO: 126, (b) amino acids 586-594 of SEQ ID NO: 126, (c) amino acids 626-650 of SEQ ID NO: 126, (d) amino acids 693-703 of SEQ ID NO: 126, (e) amino acids 704-724 of SEQ ID NO: 126, and / or (e) amino acids 780-805 of SEQ ID NO: 126, as determined by hydrogen / deuterium exchange. In other embodiments, the antibody or antigen-binding fragment thereof interacts with an amino acid sequence selected from (a) amino acids 693-703 of SEQ ID NO: 126, and / or (b) amino acids 780-805 of SEQ ID NO: 126, as determined by hydrogen / deuterium exchange. In certain embodiments, the antibody or antigen-binding fragment thereof interacts with an amino acid sequence selected from: (a) amino acids 125-133 of SEQ ID NO:126; (b) amino acids 586-594 of SEQ ID NO:126; (c) amino acids 626-650 of SEQ ID NO:126; and / or (d) amino acids 704-724 of SEQ ID NO:126, as determined by hydrogen / deuterium exchange.

[0015] In another aspect, the disclosure provides an isolated antibody or antigen-binding fragment thereof that specifically binds to transmembrane serine protease 6 (TMPRSS6) protein, wherein the antibody or antigen-binding fragment thereof interacts with residues G699, H701, D724, Q726, L727, I728, P729, L732, E735, G749, Y750, R751, K752, and N791 of human TMPRSS6 (SEQ ID NO: 126) as determined by cryo-electron microscopy structural modeling.

[0016] In certain embodiments, the antibody or antigen-binding fragment thereof further interacts with residues V490, S501, T502, C503, I504, S505, and K508 of human TMPRSS6 (SEQ ID NO: 126) as determined by cryo-electron microscopy structural modeling.

[0017] In another aspect, the disclosure provides an isolated antibody or antigen-binding fragment thereof that specifically binds to transmembrane serine protease 6 (TMPRSS6) protein, wherein the antibody or antigen-binding fragment thereof interacts with residues R597, R599, I601, D622, L710, R711, E712, G713, G714, P715, and I716 of human TMPRSS6 as determined by cryo-electron microscopy structural modeling.

[0018] In another aspect, the disclosure provides an isolated antibody or antigen-binding fragment thereof that specifically binds to a transmembrane serine protease 6 (TMPRSS6) protein, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR), wherein the HCDR3 has an amino acid sequence selected from the sequences of HCDR3 set forth in Table 1.

[0019] In certain embodiments, the LCDR3 sequence has an amino acid sequence selected from the LCDR3 sequences set forth in Table 1. In certain embodiments, the antibody or antigen-binding fragment comprises an HCDR1, HCDR2, and HCDR3 sequence set forth in Table 1. In certain embodiments, the antibody or antigen-binding fragment comprises an LCDR1, LCDR2, and LCDR3 sequence set forth in Table 1.

[0020] In certain embodiments, (a) the HCDR1 domain has an amino acid sequence selected from SEQ ID NO: 4, 24, 34, 44, 64, 84, or 102; (b) the HCDR2 domain has an amino acid sequence selected from SEQ ID NO: 6, 26, 36, 46, 66, 86, or 104; (c) the HCDR3 domain has an amino acid sequence selected from SEQ ID NO: 8, 28, 38, 48, 68, 88, or 106; (d) the LCDR1 domain has an amino acid sequence selected from SEQ ID NO: 12, 52, 72, 92, or 110; (e) the LCDR2 domain has an amino acid sequence selected from SEQ ID NO: 14, 54, or 74; and (f) the LCDR3 domain has an amino acid sequence selected from SEQ ID NO: 16, 56, 76, 94, or 112.

[0021] In certain embodiments, the three heavy chain CDRs and the three light chain CDRs comprise one of the following CDR sets: (a) SEQ ID NO:4 (HCDR1), SEQ ID NO:6 (HCDR2), SEQ ID NO:8 (HCDR3), SEQ ID NO:12 (LCDR1), SEQ ID NO:14 (LCDR2), and SEQ ID NO:16 (LCDR3); (b) SEQ ID NO:24 (HCDR1), SEQ ID NO:26 (HCDR2), SEQ ID NO:28 (HCDR3), SEQ ID NO:12 (LCDR1), SEQ ID NO:14 (LCDR2), and SEQ ID NO:16 (LCDR3); (c) SEQ ID NO:34 (HCDR1), SEQ ID NO:36 (HCDR2), SEQ ID NO:38 (HCDR3), SEQ ID NO:12 (LCDR1), SEQ ID NO:14 (LCDR2), and SEQ ID NO:16 (LCDR3); (d) SEQ ID NO:44 (HCDR1), SEQ ID NO:46 ( HCDR2), SEQ ID NO:48 (HCDR3), SEQ ID NO:52 (LCDR1), SEQ ID NO:54 (LCDR2), and SEQ ID NO:56 (LCDR3); (e) SEQ ID NO:64 (HCDR1), SEQ ID NO:66 (HCDR2), SEQ ID NO:68 (HCDR3), SEQ ID NO:72 (LCDR1), SEQ ID NO:74 (LCDR2), and SEQ ID NO:76 (LCDR3); (f) SEQ ID NO:84 (HCDR1), SEQ ID NO:86 (HCDR2), SEQ ID NO:88 (HCDR3), SEQ ID NO:92 (LCDR1), SEQ ID NO:14 (LCDR2), and SEQ ID NO:94 (LCDR3); or (g) SEQ ID NO:102 (HCDR1), SEQ ID NO:104 (HCDR2), SEQ ID NO:106 (HCDR3), SEQ ID NO:110 (LCDR1), SEQ ID NO:14 (LCDR2), and SEQ ID NO:112 (LCDR3).

[0022] In certain embodiments, the three heavy chain CDRs and the three light chain CDRs comprise the following CDR sets: SEQ ID NO: 44 (HCDR1), SEQ ID NO: 46 (HCDR2), SEQ ID NO: 48 (HCDR3), SEQ ID NO: 52 (LCDR1), SEQ ID NO: 54 (LCDR2), and SEQ ID NO: 56 (LCDR3). In other embodiments, the three heavy chain CDRs and the three light chain CDRs comprise the following CDR sets: SEQ ID NO: 102 (HCDR1), SEQ ID NO: 104 (HCDR2), SEQ ID NO: 106 (HCDR3), SEQ ID NO: 110 (LCDR1), SEQ ID NO: 14 (LCDR2), and SEQ ID NO: 112 (LCDR3).

[0023] In another aspect, the disclosure provides an antibody or antigen-binding fragment thereof that binds to TMPRSS6, the antibody or antigen-binding fragment thereof comprising three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within an HCVR and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within an LCVR, the HCVR comprising: (i) an amino acid sequence selected from SEQ ID NO: 2, 22, 32, 42, 62, 82, or 100; (ii) an amino acid sequence having at least 90% identity to an amino acid sequence selected from SEQ ID NO: 2, 22, 32, 42, 62, 82, or 100; (iii) an amino acid sequence having at least 95% identity to an amino acid sequence selected from SEQ ID NO: 2, 22, 32, 42, 62, 82, or 100; or (iv) an amino acid sequence having no more than 12 amino acid substitutions relative to a sequence selected from SEQ ID NO: 2, 22, 32, 42, 62, 82, or 100.

[0024] In certain embodiments, the LCVR comprises (a) an amino acid sequence selected from SEQ ID NO: 10, 50, 70, 90, or 108; (b) an amino acid sequence having at least 90% identity to an amino acid sequence selected from SEQ ID NO: 10, 50, 70, 90, or 108; (c) an amino acid sequence having at least 95% identity to an amino acid sequence selected from SEQ ID NO: 10, 50, 70, 90, or 108; or (d) an amino acid sequence having 10 or fewer amino acid substitutions relative to a sequence selected from SEQ ID NO: 10, 50, 70, 90, or 108.

[0025] In certain embodiments, the HCVR comprises an amino acid sequence selected from SEQ ID NO: 2, 22, 32, 42, 62, 82, or 100. In certain embodiments, the LCVR comprises an amino acid sequence selected from SEQ ID NO: 10, 50, 70, 90, or 108.

[0026] In certain embodiments, the antibody or antigen-binding fragment thereof comprises an HCVR / LCVR amino acid sequence pair selected from SEQ ID NOs: 2 / 10, 22 / 10, 32 / 10, 42 / 50, 62 / 70, 82 / 90, or 100 / 108. In certain embodiments, the antibody or antigen-binding fragment comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 42 / 50. In other embodiments, the antibody or antigen-binding fragment comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 100 / 108.

[0027] In another aspect, the disclosure provides an antibody that binds to TMPRSS6, wherein the antibody comprises a heavy chain (HC) and a light chain (LC), wherein the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 30, 40, 58, 78, 96, and 114.

[0028] In certain embodiments, the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 60, 80, 98 and 116. In certain embodiments, the antibody comprises an HC / LC amino acid sequence pair selected from SEQ ID NOs: 18 / 20, 30 / 20, 40 / 20, 58 / 60, 78 / 80, 96 / 98 or 114 / 116. In certain embodiments, the antibody comprises a 58 / 60 HC / LC amino acid sequence pair. In other embodiments, the antibody comprises a 114 / 116 HC / LC amino acid sequence pair.

[0029] In certain embodiments, the antibodies or antigen-binding fragments of the present disclosure have one or more properties selected from the following: (a) are fully human monoclonal antibodies or antigen-binding fragments thereof; (b) have a dissociation constant (K) of less than about 21.2 nM for human TMPRSS6 at 25° C. and 37° C. as measured by a surface plasmon resonance assay. D ) binds to monkey TMPRSS6 with a K of less than about 25.7 nM at 25° C. and 37° C. as measured by surface plasmon resonance assays; D (d) binds to mouse TMPRSS6 with a K of less than about 703 nM at 25° C. and 37° C. as measured by surface plasmon resonance assays. D(e) binds to cells expressing human TMPRSS6 and inhibits EC 50 (f) binds to cells expressing monkey TMPRSS6 and has an EC 50 (g) binds to cells expressing mouse TMPRSS6 and has an EC 50 (h) inhibits protease-dependent release of cell surface hemojuvelin in the presence of human TMPRSS6 with a percent inhibition of greater than about 90%, e.g., inhibits human TMPRSS6; (i) inhibits protease-dependent release of cell surface hemojuvelin in the presence of human TMPRSS6, e.g., inhibits human TMPRSS6 and has an IC of less than about 200 pM. 50 (j) inhibits protease-dependent release of cell surface hemojuvelin in the presence of mouse TMPRSS6 with a percent inhibition of greater than about 42%, e.g., inhibits mouse TMPRSS6; (k) inhibits protease-dependent release of cell surface hemojuvelin in the presence of mouse TMPRSS6 with an IC of less than about 274 pM, e.g., inhibits mouse TMPRSS6. 50 (l) inhibits human TMPRSS6 with a percent inhibition of greater than about 65% at 25° C. and 37° C.; (m) inhibits human TMPRSS6 with an IC of less than about 10 nM at 25° C. and 37° C. 50 (n) inhibits monkey TMPRSS6 with a percent inhibition of greater than about 66% at 25° C. and 37° C.; (o) inhibits monkey TMPRSS6 with an IC of less than about 50 nM at 25° C. and 37° C. 50 (p) inhibits mouse TMPRSS6 with a percent inhibition of greater than about 58% at 25° C. and 37° C.; (q) inhibits mouse TMPRSS6 with an IC of less than about 35 nM at 25° C. and 37° C. 50(r) when administered to a subject in need thereof, decreases the serum iron concentration in the subject, (s) when administered to a subject in need thereof, increases the serum hepcidin concentration in the subject, (t) when administered to a subject in need thereof, increases the level of mature red blood cells in the subject, (u) when administered to a subject in need thereof, increases the level of mature red blood cells in the spleen and / or bone marrow of the subject, (v) when administered to a subject in need thereof, increases the hemoglobin concentration in the subject, or (w) when administered to a subject in need thereof, decreases the transferrin saturation level in the subject. In certain embodiments, the antibody is a fully human monoclonal antibody.

[0030] In another aspect, the disclosure provides an antibody or antigen-binding fragment thereof that competes with an antibody or antigen-binding fragment thereof disclosed herein for binding to TMPRSS6.

[0031] In another aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to the same epitope as an antibody or antigen-binding fragment thereof disclosed herein.

[0032] In another aspect, the disclosure provides a pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof that binds to TMPRSS6, as disclosed herein, and a pharma- ceutically acceptable carrier or diluent.

[0033] In another aspect, the disclosure provides an isolated polynucleotide molecule comprising a polynucleotide sequence encoding the HCVR of an antibody or antigen-binding fragment thereof disclosed herein.

[0034] In another aspect, the present disclosure provides an isolated polynucleotide molecule comprising a polynucleotide sequence encoding the LCVR of an antibody or antigen-binding fragment thereof disclosed herein.

[0035] In another aspect, the disclosure provides a vector comprising a polynucleotide sequence disclosed herein.

[0036] In another aspect, the present disclosure provides a set of vectors comprising a first vector comprising a polynucleotide sequence encoding the HCVR of an antibody or antigen-binding fragment thereof disclosed herein, and a second vector comprising a polynucleotide sequence encoding the LCVR of an antibody or antigen-binding fragment thereof disclosed herein.

[0037] In another aspect, the disclosure provides a host cell comprising a vector or set of vectors disclosed herein.

[0038] In another aspect, the disclosure provides a method of producing an antibody or antigen-binding fragment thereof that specifically binds to TMPRSS6, comprising culturing a host cell of the disclosure under conditions that allow for production of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof so produced. In certain embodiments, the method further comprises formulating the antibody or antigen-binding fragment thereof as a pharmaceutical composition comprising a pharma- ceutically acceptable carrier.

[0039] In another aspect, the present disclosure provides a method for treating, preventing, or ameliorating at least one symptom or sign of a disease or disorder associated with iron overload, comprising administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof of the present disclosure.

[0040] In certain embodiments, the disease or disorder is congenital dyserythroplastic anemia, Diamond-Blackfan anemia, alpha thalassemia, beta thalassemia, transfusion-dependent hemolytic anemia, myelodysplastic syndrome, sickle cell disease, polycythemia vera, hereditary hemochromatosis, or chronic liver disease. In certain embodiments, the disease or disorder is beta thalassemia. In certain embodiments, the disease or disorder is beta thalassemia major or beta thalassemia intermedia. In certain embodiments, the disease or disorder is myelodysplastic syndrome. In certain embodiments, the disease or disorder is myelodysplastic syndrome with ringed sideroblasts. In certain embodiments, the disease or disorder is transfusion-dependent hemolytic anemia. In certain embodiments, the disease or disorder is transfusion-dependent hemolytic anemia due to pyruvate kinase deficiency or sideroblastic transfusion-dependent hemolytic anemia. In certain embodiments, the disease or disorder is chronic liver disease. In certain embodiments, the disease or disorder is alcohol-related chronic liver disease, hepatitis C, or autoimmune hepatitis.

[0041] In certain embodiments, the pharmaceutical composition is administered prophylactically or therapeutically to a subject in need thereof, hi other embodiments, the pharmaceutical composition is administered subcutaneously, intravenously, intradermally, intraperitoneally, or intramuscularly.

[0042] Other embodiments will become apparent from a review of the following detailed description. [Brief description of the drawings]

[0043] [Figure 1] 1 is a schematic diagram of the study design for the mouse forced running study in Example 15. [Diagram 2]The effect of REGN7999 treatment on running distance (A) and lactate production over running distance (B) in Hbbth3 / + mice. Control animals were wild-type (WT) C57BL / 6 mice or Hbbth3 / + mice treated with an isotype control antibody (REGN1945). Note: All data in the figures are presented as mean ± standard deviation (SD), and P < 0.05 indicates a statistically significant difference under one-way ANOVA and Tukey post-hoc multiple comparison test. [Diagram 3] Shown is the running distance of wild type (WT) C57BL / 6 mice treated with either REGN7999 or an isotype control antibody (REGN1945). [Figure 4] Serum hepcidin (A), serum iron (B), and liver iron (C) concentrations in Hbbth3 / + mice treated with either REGN7999 or Acvr2b(L79D)-Fc for 8 weeks are shown. Control animals were wild-type (WT) C57BL / 6 mice or Hbbth3 / + mice treated with an isotype control antibody (REGN1945). [Diagram 5] Average body weight (g) of WT or Hbbth3 / + mice after 8 weeks of treatment with REGN7999, Acvr2b(L79D)-Fc, or isotype control antibody (REGN1945) is shown. WT mice treated with isotype control: circles, solid black line. Hbbth3 / + mice treated with isotype control: squares, solid grey line. Hbbth3 / + mice treated with REGN7999: triangles (upwards), dotted black line. Hbbth3 / + mice treated with Acvr2b(L79D)-Fc: triangles (downwards), dotted grey line. [Figure 6] Shown is the average spleen weight (g) of WT or Hbbth3 / + mice after 8 weeks of treatment with REGN7999, Acvr2b(L79D)-Fc, or isotype control antibody (REGN1945). [Figure 7]Shown are the amounts of mature RBCs (%CD44lowTer119+ cells) in spleens from WT or Hbbth3 / + mice 8 weeks after treatment with REGN7999, Acvr2b(L79D)-Fc, or isotype control antibody (REGN1945). [Figure 8] Shown are the amounts of reticulocytes (% CD44highTer119+ cells) from WT or Hbbth3 / + mice after 8 weeks of treatment with REGN7999, Acvr2b(L79D)-Fc, or isotype control antibody (REGN1945). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] Before the present methods are described, it is understood that this disclosure is not limited to particular methods and experimental conditions, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the relevant art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosed embodiments, the preferred methods and materials are described herein. All publications mentioned herein are incorporated by reference in their entirety.

[0046] definition The term "TMPRSS6," also known as "matriptase-2" or "MTP-2," refers to transmembrane serine protease 6. TMPRSS6 consists of a short N-terminal intracellular region (residues 1-55 of human TMPRSS6 (SEQ ID NO:126)) followed by a single transmembrane domain (residues 56-76 of SEQ ID NO:126), a single SEA (sea urchin sperm protein, enteropeptidase, and agrin) domain (residues 84-209 of SEQ ID NO:126), two CUB (Cls / Clr, sea urchin embryonic growth factor, and bone morphogenetic protein) domains (residues 85-209 of SEQ ID NO:126), and a 5'-terminal cytoplasmic domain (residues 86-209 of SEQ ID NO:126) that is connected to two CUB (Cls / Clr, sea urchin embryonic growth factor, and bone morphogenetic protein) domains. It is a type II transmembrane serine protease composed of a BMP-1 domain (residues 213-336 of SEQ ID NO:126 for CUB domain 1 and residues 335-452 of SEQ ID NO:126 for CUB domain 2), three LDLRa (low density lipoprotein receptor, class A) repeat domains (residues 457-489 of SEQ ID NO:126 for LDLRa domain 1, residues 492-526 of SEQ ID NO:126 for LDLRa domain 2, and residues 530-567 of SEQ ID NO:126 for LDLRa domain 3), and a C-terminal catalytic serine protease domain (residues 577-811 of SEQ ID NO:126). The catalytic serine protease domain contains a catalytic triad of histidine (residue 617 of human TMPRSS6 (SEQ ID NO:126)), aspartic acid (residue 668 of SEQ ID NO:126), and serine residues (residue 762 of SEQ ID NO:126) (Lee, Acta Haematol. (2009) 122(2-3):87-96; Ramsay et al., Front. Biosci. (2008) 13:569-579). TMPRSS6 has been shown to play a role in iron homeostasis by acting as a negative regulator of hepcidin production through cleavage of the BMP co-receptor hemojuvelin (HJV) (Dion et al., Scientific Reports (2018) 8:12562).The amino acid sequence of the full-length human TMPRSS6 protein (isoform 2) is exemplified by the 811 amino acid sequence provided in UniProtKB / Swiss-Prot under accession number Q8IU80, corresponding to NCBI accession number NP_705837.1 (TMPS6_human; SEQ ID NO: 126). Human TMPRSS6 protein is encoded by the nucleic acid sequence represented by the NCBI reference sequence NM_153609.3 (SEQ ID NO: 127). The amino acid sequence of the full-length cynomolgus TMPRSS6 protein is exemplified by the amino acid sequence provided in UniProtKB / Swiss-Prot under accession number A0A2K5VAP0, corresponding to NCBI accession number XP_005567441.1 (A0A2K5VAP0_MACFA; SEQ ID NO: 128). Cynomolgus TMPRSS6 protein is encoded by a nucleic acid sequence represented by NCBI Reference Sequence XM_005567384 (SEQ ID NO: 129). The amino acid sequence of full-length mouse TMPRSS6 protein is exemplified by the amino acid sequence provided in UniProtKB / Swiss-Prot under Accession No. Q9DBI0, corresponding to NCBI Accession No. NP_082178.2 (TMPS6_mouse; SEQ ID NO: 130). Mouse TMPRSS6 protein is encoded by a nucleic acid sequence represented by NCBI Reference Sequence NM_027902.2 (SEQ ID NO: 131). The term "TMPRSS6" includes recombinant TMPRSS6 protein or fragments thereof. Such term also encompasses TMPRSS6 protein or fragments thereof (e.g., SEQ ID NOs: 117-119) linked to, for example, a histidine tag, mouse or human Fc, or a signal sequence such as ROR1.

[0047] The term "about," when used in reference to a particular stated numerical value, means that the value may vary by up to 1% from the stated value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, ... 100.7, 100.8, 100.9).

[0048] The term "antibody," as used herein, is intended to refer to an immunoglobulin molecule (i.e., an "intact antibody molecule") that is composed of four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM) or antigen-binding fragments thereof. Each heavy chain contains a heavy chain variable region ("HCVR" or "V H ") and the heavy chain constant region (domain C H 1. C H 2 and C H Each light chain is composed of a light chain variable region ("LCVR" or "V L ) and light chain constant region (C L ) is composed of V H and V L The regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). H and V L is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the present disclosure, the FRs of the antibody (or antigen-binding fragment thereof) may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on the parallel analysis of two or more CDRs.

[0049] Substitution of one or more CDR residues or omission of one or more CDRs is also possible. Antibodies are described in the scientific literature, according to which one or two CDRs can be omitted for binding. Padlan et al. (1995, FASEB J. 9:133-139) analyzed the contact regions between antibodies and their antigens based on published crystal structures and concluded that only about one-fifth to one-third of the CDR residues actually contact the antigen. Padlan also found many antibodies in which one or two CDRs have no amino acids that contact the antigen (see also Vajdos et al. (2002) J Mol Biol 320:415-428).

[0050] CDR residues that do not contact the antigen can be identified based on previous studies, by molecular modeling and / or empirically, from regions of the Kabat CDRs that are outside the Chothia CDRs (e.g., residues H60-H65 of CDRH2 are often unnecessary). When a CDR or its residue(s) is omitted, it is usually replaced with an amino acid occupying the corresponding position in another human antibody sequence or a consensus of such sequences. The positions for substitution within the CDR and the amino acids to substitute can also be selected empirically. Empirical substitutions can be conservative or non-conservative.

[0051] The fully human anti-TMPRSS6 monoclonal antibodies disclosed herein may contain one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present disclosure includes antibodies, and antigen-binding fragments thereof, derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue(s) in the germline sequence from which the antibody was derived, or to the corresponding residue(s) in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as "germline mutations"). Starting with the heavy and light chain variable region sequences disclosed herein, one of skill in the art can readily generate numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, V H Domain and / or V LAll of the framework and / or CDR residues in the domains are backmutated to the residue found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are backmutated to the original germline sequence, e.g., only the mutated residues found in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only the mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residue(s) are mutated to the corresponding residue(s) in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies disclosed herein may contain any combination of two or more germline mutations in the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residue in a particular germline sequence, while other specific residues that differ from the original germline sequence are either maintained or mutated to the corresponding residue in a different germline sequence. Once antibodies and antigen-binding fragments containing one or more germline mutations are obtained, they can be readily tested for one or more desired properties, e.g., improved binding specificity, increased binding affinity, improved or enhanced competitive biological properties, reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained by this general method are encompassed within the present disclosure.

[0052] Also disclosed herein are fully human anti-TMPRSS6 monoclonal antibodies that include variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the disclosure includes anti-TMPRSS6 antibodies having HCVR, LCVR, and / or CDR amino acid sequences that include, for example, 10 or less, 8 or less, 6 or less, 4 or less, etc., conservative amino acid substitutions compared to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0053] The term "human antibody", or "fully human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human mAbs disclosed herein may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example, within the CDRs, particularly within CDR3. However, the term "human antibody", or "fully human antibody", as used herein, is not intended to include mAbs in which CDR sequences derived from the germline of another mammal (e.g., mouse) have been grafted onto human FR sequences. Such terms include antibodies recombinantly produced in non-human mammals or in the cells of non-human mammals. The term is not intended to include antibodies isolated from or generated in a human subject.

[0054] The term "recombinant" as used herein refers to antibodies or antigen-binding fragments thereof that are made, expressed, isolated or obtained by techniques or methods known in the art as recombinant DNA technology, including, for example, DNA splicing and transgenic expression. Such terms refer to antibodies that are expressed in a non-human mammalian (including a transgenic non-human mammalian, e.g., a transgenic mouse) or cellular (e.g., CHO cell) expression system or isolated from a recombinant combinatorial human antibody library.

[0055] The terms "specifically bind" or "specifically bind to" and the like mean that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is at least about 1×10 -8 M or less equilibrium dissociation constant (e.g., smaller K DThe binding of TMPRSS6 can be characterized by the binding of the two molecules to each other (wherein a stronger binding indicates a stronger binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. As described herein, an antibody has been identified by surface plasmon resonance, e.g., BIACORE™, that specifically binds to TMPRSS6. Furthermore, a multispecific antibody that binds to one domain of TMPRSS6 and one or more additional antigens, or a bispecific antibody that binds to two different regions of TMPRSS6, is nevertheless considered an antibody that "specifically binds" as used herein.

[0056] The term "high affinity" antibody refers to an antibody that has a K D Binding affinity for TMPRSS6 expressed as at least 10 -8 M, preferably 10 -9 M, more preferably 10 -10 M, and even more preferably 10 -11 M.

[0057] The term "low dissociation rate", "Koff" or "kd" refers to a low dissociation rate of 1×10 as determined by surface plasmon resonance, e.g., BIACORE™. -3 s -1 or less, preferably 1×10 -4 s -1 It means an antibody that dissociates from TMPRSS6 with a rate constant equal to or lower than that.

[0058] The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically derived, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. The term "antigen-binding fragment" of an antibody, or "antibody fragment," as used herein, refers to one or more fragments of an antibody that retain the ability to bind to a TMPRSS6 protein.

[0059] In specific embodiments, the antibodies or antibody fragments of the present disclosure may be conjugated to a moiety, such as a ligand or therapeutic moiety (an "immunoconjugate"), a second anti-TMPRSS6 antibody, or any other therapeutic moiety useful for treating a disease or disorder associated with TMPRSS6.

[0060] An "isolated antibody," as used herein, is intended to refer to an antibody that is substantially free of other antibodies (Abs) having different antigen specificities (e.g., an isolated antibody that specifically binds to TMPRSS6 or a fragment thereof is substantially free of Abs that specifically bind to antigens other than TMPRSS6).

[0061] The term "surface plasmon resonance," as used herein, refers to an optical phenomenon that allows analysis of real-time biomolecular interactions by detecting changes in protein concentration within a biosensor substrate, for example using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ).

[0062] "K D The term "as used herein" is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

[0063] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site (known as a paratope) on the variable region of an antibody molecule. A single antigen may have multiple epitopes. Thus, different antibodies may bind to different regions on an antigen and may have different biological effects. The term "epitope" also refers to the site on an antigen to which B cells and / or T cells respond. It also refers to the region of the antigen to which an antibody binds. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and have residues that directly contribute to the affinity of the interaction. Epitopes may also be conformational, i.e., composed of non-linear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments may have specific three-dimensional structural characteristics, and / or specific charge characteristics.

[0064] In some embodiments, the antibodies and antigen-binding fragments disclosed herein comprise an epitope or a portion of an epitope including G699, H701, D724, Q726, L727, I728, P729, L732, E735, G749, Y750, R751, K752, and N791 of human TMPRSS6 (NP_705837.1; SEQ ID NO: 126). In some embodiments, the antibodies and antigen-binding fragments disclosed herein comprise an epitope or a portion of an epitope including V490, S501, T502, C503, I504, S505, and K508 of human TMPRSS6 (NP_705837.1; SEQ ID NO: 126). In some embodiments, the antibodies and antigen-binding fragments disclosed herein comprise an epitope comprising two portions of human TMPRSS6: a first portion comprising G699, H701, D724, Q726, L727, I728, P729, L732, E735, G749, Y750, R751, K752, and N791 of human TMPRSS6, and a second portion comprising V490, S501, T502, C503, I504, S505, and K508 of human TMPRSS6 (NP_705837.1; SEQ ID NO: 126).

[0065] In other embodiments, the antibodies and antigen-binding fragments disclosed herein comprise an epitope or a portion of an epitope including R597, R599, I601, D622, L710, R711, E712, G713, G714, P715, and I716 of human TMPRSS6 (NP_705837.1; SEQ ID NO: 126).

[0066] The term "cross-compete" as used herein means that an antibody or antigen-binding fragment thereof binds to an antigen and inhibits or blocks the binding of another antibody or antigen-binding fragment thereof. Such term also includes bidirectional competition between two antibodies, i.e., a first antibody that binds and blocks the binding of a second antibody, and vice versa. In certain embodiments, the first antibody and the second antibody may bind to the same epitope. Alternatively, the first and second antibodies may bind to different but overlapping epitopes such that the binding of one inhibits or blocks the binding of the second antibody, for example, by steric hindrance. Cross-competition between antibodies can be measured by methods known in the art, for example, real-time label-free biolayer interferometry. Cross-competition between two antibodies can be expressed as the binding of the second antibody being less than the background signal due to self-self binding (the first and second antibodies are the same antibody). Cross-competition between two antibodies can be expressed, for example, as the % binding of a second antibody below the baseline self-self background binding (where the first and second antibodies are the same antibody).

[0067] The term "substantial identity" or "substantially identical," when referring to a nucleic acid or a fragment thereof, indicates that when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions, as discussed below, there is nucleotide sequence identity of at least about 90%, more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases as measured by any well-known sequence identity algorithm, such as FASTA, BLAST, or GAP. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0068] The term "substantial similarity" or "substantially similar" as applied to polypeptides means that two peptide sequences share at least 90% sequence identity, and even more preferably at least 95%, 98% or 99% sequence identity, when optimally aligned, such as by the GAP or BESTFIT programs using default gap weights. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially change the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331, incorporated herein by reference. Examples of groups of amino acids with side chains of similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine, 2) aliphatic hydroxyl side chains: serine and threonine, 3) amide-containing side chains: asparagine and glutamine, 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan, 5) basic side chains: lysine, arginine, and histidine, 6) acidic side chains: aspartic acid and glutamic acid, and 7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Instead, a conservative replacement is any change that has a positive value in the PAM250 log-likelihood matrix, as disclosed in Gonnet et al. (1992) Science 256:1443 45, which is incorporated herein by reference. A "moderately conservative" replacement is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0069] Polypeptide sequence similarity is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For example, GCG software includes programs such as GAP and BESTFIT, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species, or between a wild-type protein and its mutant protein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using the program FASTA, included in GCG version 6.1, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment and percent sequence identity of the best overlapping regions between the query and search sequences (Pearson (2000) supra). Another preferred algorithm for comparing a sequence against a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, with default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and (1997) Nucleic Acids Res. 25:3389-3402, each of which is incorporated herein by reference.

[0070] The phrase "therapeutically effective amount" means an amount that produces the desired effect for which it is administered. The exact amount will depend on the purpose of the treatment and can be ascertained by one skilled in the art using known procedures (see, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0071] As used herein, the term "subject" refers to an animal, preferably a mammal, more preferably a human, in need of amelioration, prevention and / or treatment of a disease or disorder associated with TMPRSS6, such as beta thalassemia or myelodysplastic syndrome. Such term includes human subjects having or at risk of having such a disease or disorder.

[0072] As used herein, the terms "treat", "treating" or "treatment" refer to a reduction or amelioration of the severity of at least one symptom or sign of a disease or disorder associated with TMPRSS6 resulting from administration of a therapeutic agent, such as an antibody or antigen-binding fragment disclosed herein, to a subject in need thereof. Such terms include inhibition of progression of the disease or inhibition of worsening of symptoms / signs. Such terms also include a favorable prognosis of the disease, i.e., the subject may be disease-free or have a remission of the disease upon administration of a therapeutic agent, such as an antibody or antigen-binding fragment disclosed herein. The therapeutic agent may be administered to the subject in a therapeutic dose.

[0073] The terms "prevent," "preventing," or "prevention" refer to the inhibition of the onset of a disease or disorder associated with TMPRSS6, or any symptom or sign of such a disease or disorder, upon administration of an antibody or antigen-binding fragment disclosed herein.

[0074] Antigen-binding fragment of an antibody Unless otherwise specifically indicated, the term "antibody", as used herein, shall be understood to encompass an antibody molecule comprising two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., a "complete antibody molecule") as well as antigen-binding fragments thereof. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically derived, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. The term "antigen-binding fragment" of an antibody, or "antibody fragment", as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to a TMPRSS6 protein. Antibody fragments may include Fab fragments, F(ab')2 fragments, Fv fragments, dAb fragments, fragments containing CDRs, or isolated CDRs. Antigen-binding fragments of antibodies may be derived, for example, from complete antibody molecules using any suitable standard technique, such as proteolytic digestion, or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding the variable and (optionally) constant domains of the antibody. Such DNA is known and / or readily available, e.g., from commercial sources, DNA libraries (e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and chemically engineered or may be engineered, e.g., to position one or more variable and / or constant domains in a suitable configuration, or by using molecular biology techniques to introduce codons, create cysteine ​​residues, modify, add or delete amino acids, etc.

[0075] Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single chain Fv (scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable regions (e.g., isolated complementarity determining regions (CDRs) such as CDR3 peptides) of an antibody or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark IgNAR variable domains, are also encompassed within the term "antigen-binding fragment" as used herein.

[0076] Antigen-binding fragments of antibodies typically contain at least one variable domain. The variable domain may be of any size or amino acid composition and generally contains at least one CDR adjacent to or in frame with one or more framework sequences. L Domain and associated V H For antigen-binding fragments containing domains, V H Domain and V L The domains can be positioned in any suitable arrangement relative to each other. For example, the variable region is a dimer and the V H -V H , V H -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may contain a dimer of monomeric V H Domain or V L It may contain domains.

[0077] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in the antigen-binding fragments of an antibody disclosed herein include: (i) V H -C H 1, (ii) V H -C H 2. (iii) V H -C H 3. (iv) V H -C H 1-C H 2. (v) V H -C H 1-C H 2-C H 3. (vi) V H -C H 2-C H 3. (vii) V H -C L , (viii) V L -C H 1, (ix) V L -C H 2. (x)V L -C H 3. (xi) V L -C H 1-C H 2. (xii) V L -C H 1-C H 2-C H 3. (xiii) V L -C H 2-C H 3, and (xiv) V L -C LIn any configuration of the variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which provide a flexible or semi-flexible linkage between adjacent variable and / or constant domains within a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies disclosed herein may be linked to each other and / or to one or more monomeric V H Domain or V L The variable domain and constant domain may comprise homodimers or heterodimers (or other multimers) of any of the above listed configurations of variable and constant domains in which the domains are non-covalently associated (e.g., by disulfide bond(s)).

[0078] As with intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically contain at least two different variable domains, each capable of specifically binding to a separate antigen or to a different epitope of the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in conjunction with the antigen-binding fragments of antibodies disclosed herein using routine techniques available in the art.

[0079] Preparation of human antibodies Methods for producing human antibodies in transgenic mice are known in the art. Any such known method can be used in the context of the present disclosure to produce human antibodies that specifically bind to TMPRSS6.

[0080] To generate antibodies against TMPRSS6 protein, an immunogen comprising any one of the following may be used. In certain embodiments, the antibodies disclosed herein are obtained from mice immunized with full-length native TMPRSS6 protein (see, for example, UniProtKB / Swiss-Prot Accession No. Q8IU80) or DNA encoding the protein or a fragment thereof. Alternatively, the protein or a fragment thereof may be produced and modified using standard biochemical techniques and used as an immunogen.

[0081] In some embodiments, the immunogen can be a recombinant TMPRSS6 protein or a fragment thereof expressed in E. coli or any other eukaryotic or mammalian cell, such as Chinese Hamster Ovary (CHO) cell (e.g., SEQ ID NOs: 117-119).

[0082] Using VELOCIMMUNE® technology (see, e.g., US 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method for producing monoclonal antibodies, a high affinity chimeric antibody against TMPRSS6 is first isolated with a human variable region and a mouse constant region. VELOCIMMUNE® technology involves the creation of a transgenic mouse with a genome that includes human heavy and light chain variable regions operably linked to endogenous mouse constant region sites such that the mouse produces antibodies containing the human variable region and the mouse constant region in response to antigenic stimulation. DNA encoding the antibody heavy and light chain variable regions is isolated and operably linked to DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in a cell capable of expressing a fully human antibody.

[0083] Generally, VELOCIMMUNE® mice are administered an antigen of interest and lymphoid cells (such as B cells) are collected from the mice that express antibodies. Lymphoid cells may be fused with a myeloma cell line to prepare immortal hybridoma cell lines, which are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the heavy and light chain variable regions may be isolated and linked to constant regions of the heavy and light chains of the desired isotype. Such antibody proteins may be produced in cells, such as CHO cells. Alternatively, DNA encoding the antigen-specific chimeric antibody or the light and heavy chain variable domains may be isolated directly from antigen-specific lymphocytes.

[0084] First, a high affinity chimeric antibody is isolated with human variable regions and mouse constant regions. As in the experimental section below, the antibodies are characterized and selected for desirable characteristics including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with the desired human constant regions to generate fully human antibodies as disclosed herein, e.g., wild-type or modified IgG1 or IgG4. The constant regions selected may vary depending on the particular application, but the characteristics of high affinity antigen binding and target specificity reside in the variable regions.

[0085] biological equivalent The anti-TMPRSS6 antibodies and antibody fragments disclosed herein include proteins having amino acid sequences that differ from those of the described antibodies but that retain the ability to bind to TMPRSS6 protein. Such variant antibodies and antibody fragments contain one or more amino acid additions, deletions, or substitutions when compared to the parent sequence, but exhibit essentially the same biological activity as the described antibody. Similarly, the DNA sequences encoding the antibodies disclosed herein include sequences that contain one or more nucleotide additions, deletions, or substitutions when compared to the sequences of the present disclosure, but encode antibodies or antibody fragments that are essentially biologically equivalent to the antibodies or antibody fragments disclosed herein.

[0086] Two antigen-binding proteins, or antibodies, are considered bioequivalent, for example, if they are pharmaceutical equivalents or pharmaceutical substitutes that do not exhibit significant differences in the rate and extent of absorption when administered at the same molar dose under similar experimental conditions, either in single or multiple doses. Some antibodies may be considered equivalents or pharmaceutical substitutes if they are equivalent in their extent of absorption but not in their rate of absorption, and furthermore, such differences in absorption rates may be considered bioequivalent because they are intentional and reflected in the labeling, are not essential to achieving effective body drug concentrations, e.g., during chronic use, and are not considered medically significant in the case of the particular pharmaceutical product being tested.

[0087] In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, or potency.

[0088] In one embodiment, two antigen binding proteins are bioequivalent if a patient is able to switch one or more times between the reference product and the biological product without a predicted increase in the risk of adverse effects, such as clinically important changes in immunogenicity or reduced efficacy, compared to continuing treatment without such switches.

[0089] In one embodiment, two antigen binding proteins are bioequivalent if they both act by a common mechanism(s) of action and to a degree known for such mechanism(s) upon the condition(s) of use.

[0090] Bioequivalence may be demonstrated by in vivo and / or in vitro methods. Assessment of bioequivalence may include, for example, (a) in vivo studies in humans or other mammals that measure the concentration of the antibody or its metabolites in blood, plasma, serum, or other biological fluid as a function of time, (b) in vitro studies that correlate with and are reasonably predictive of human in vivo bioavailability data, (c) in vivo studies in humans or other mammals that measure the relevant acute pharmacological effects of the antibody (or its target) as a function of time, and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability or bioequivalence of the antibody.

[0091] Biologically equivalent variants of the antibodies of the present disclosure can be constructed, for example, by making various substitutions of residues or sequences, or by deleting terminal or internal residues or sequences that are not required for biological activity.For example, cysteine ​​residues that are not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges during renaturation.In other contexts, biologically equivalent antibodies can include antibody variants that contain amino acid changes that change the glycosylation properties of the antibody, for example, those that contain mutations that eliminate or remove glycosylation.

[0092] Anti-TMPRSS6 antibodies, including Fc variants The antibodies referred to herein typically have fully human variable regions, but may have human or mouse constant regions. As will be appreciated by those skilled in the art, an antibody with a particular Fc isotype can be converted to an antibody with a different Fc isotype (e.g., an antibody with a mouse IgG1 Fc can be converted to an antibody with a human IgG4, etc.), but in each case, the variable domains (including the CDRs) indicated by the numerical identifiers in Table 1 remain the same, and the binding characteristics to the antigen are expected to be the same or substantially similar regardless of the nature of the Fc domain. In certain embodiments, the antibody comprises a human IgG4 Fc. In one embodiment, the human IgG4 Fc comprises a serine to proline mutation in the hinge region (S108P) to promote dimer stabilization.

[0093] According to certain embodiments disclosed herein, there are provided anti-TMPRSS6 antibodies comprising an Fc domain that includes one or more mutations that enhance or attenuate binding of the antibody to the FcRn receptor, e.g., at acidic pH compared to neutral pH. For example, the disclosure includes an Fc domain that includes a C H 2nd Area or C HIncluded are anti-TMPRSS6 antibodies that contain mutations in three regions, where the mutation(s) increase the affinity of the Fc domain for FcRn in acidic environments (e.g., within endosomes, where the pH ranges from about 5.5 to about 6.0). Such mutations can result in an increased serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T); or at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., A, W, H, F or Y [N434A, N434W, N434H, N434F or N434Y]); or at positions 250 and / or 428; or at positions 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); and 307 and / or 308 modifications (e.g., 308F or 308P). In yet another embodiment, the modifications include 265A (e.g., D265A) and / or 297A (e.g., N297A) modifications.

[0094] For example, the disclosure includes anti-TMPRSS6 antibodies that include an Fc domain that includes one or more pairs or groups of mutations selected from: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); 257I and 311I (e.g., P257I and Q311I); 257I and 434H (e.g., P257I and N434H); 376V and 434H (e.g., D376V and N434H); 307A, 380A and 434A (e.g., T307A, E380A and N434A); and 433K and 434F (e.g., H433K and N434F). All possible combinations of the aforementioned Fc domain mutations and other mutations in the antibody variable domains disclosed herein are contemplated within the scope of this disclosure.

[0095] Also referred to herein is the chimeric heavy chain constant (C H Also disclosed is an anti-TMPRSS6 antibody comprising a chimeric C H The region is C H For example, the antibodies disclosed herein include segments derived from C regions derived from human IgG1, human IgG2 or human IgG4 molecules. H C derived from a human IgG1 molecule, a human IgG2 molecule, or a human IgG4 molecule in combination with some or all of the three domains. H Chimera C containing all or part of the 2 domains H According to certain embodiments, the antibodies disclosed herein may comprise a chimeric C region having a chimeric hinge region. HFor example, the chimeric hinge may comprise an "upper hinge" amino acid sequence (amino acid residues 216-227 according to EU numbering) derived from a human IgG1, human IgG2 or human IgG4 hinge region in combination with a "lower hinge" sequence (amino acid residues 228-236 according to EU numbering) derived from a human IgG1, human IgG2 or human IgG4 hinge region. According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1 or human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge. The chimeric hinge as described herein may be H Antibodies comprising the region may, in certain embodiments, exhibit modified Fc effector functions without adversely affecting the therapeutic or pharmacokinetic properties of the antibody (see, e.g., U.S. Patent Application Publication No. 2014 / 0243504, the disclosure of which is incorporated herein by reference in its entirety).

[0096] Biological properties of antibodies and their antigen-binding fragments Generally, the antibodies or antigen-binding fragments thereof of the present disclosure function by binding to the TMPRSS6 protein and inhibiting its activity (e.g., enzymatic activity). In some embodiments, the TMPRSS6 activity that is inhibited is proteolytic activity.

[0097] Disclosed herein are antibodies and antigen-binding fragments thereof that bind to TMPRSS6 protein (i.e., anti-TMPRSS6 antibodies or antigen-binding fragments thereof). In certain embodiments, the anti-TMPRSS6 antibodies or antigen-binding fragments thereof bind to the catalytic domain of TMPRSS6. In certain embodiments, the anti-TMPRSS6 antibodies or antigen-binding fragments thereof bind to the catalytic domain of TMPRSS6 but do not bind to the catalytic triad of TMPRSS6. Also disclosed herein are antibodies and antigen-binding fragments thereof that bind at a site located within LDLRa domain 2 of TMPRSS6 (e.g., residues 492-526 of human TMPRSS6 (SEQ ID NO: 126)). In some embodiments, the antibodies and antigen-binding fragments thereof may bind to or interact with multiple sites on TMPRSS6. For example, an antibody or antigen-binding fragment thereof that binds to a site within the catalytic domain of TMPRSS6 may also bind at a site (e.g., a secondary site) contained within LDLRa domain 2 of TMPRSS6.

[0098] Further disclosed herein are antibodies and antigen-binding fragments thereof that bind to the catalytic domain of TMPRSS6 (e.g., residues 577-811 of human TMPRSS6 (SEQ ID NO: 126)), where the binding does not directly occlude the catalytic triad of TMPRSS6. Without being bound by theory, if the binding of an anti-TMPRSS6 antibody or antigen-binding fragment thereof does not directly occlude the catalytic triad of TMPRSS6, TMPRSS6 may retain its ability to bind to a substrate, e.g., hemojuvelin (HJV). For example, in certain embodiments, an anti-TMPRSS6 antibody or antigen-binding fragment thereof binds to TMPRSS6 and maintains TMPRSS6 in an inactive conformation that can bind to but cannot cleave a TMPRSS6 substrate. In certain embodiments, an anti-TMPRSS6 antibody or antigen-binding fragment thereof binds to TMPRSS6 and maintains TMPRSS6 in an inactive conformation that can bind to but cannot cleave hemojuvelin. Such anti-TMPRSS6 antibodies and antigen-binding fragments thereof may mediate allosteric regulation of TMPRSS6. In certain embodiments, the anti-TMPRSS6 antibodies or antigen-binding fragments thereof mediate allosteric inhibition of TMPRSS6. Also disclosed herein are antibodies and antigen-binding fragments thereof that bind to human TMPRSS6 protein and are allosteric inhibitors of TMPRSS6.

[0099] Also disclosed herein are antibodies and antigen-binding fragments thereof that bind to the catalytic domain of TMPRSS6, which binding directly occludes the catalytic triad of TMPRSS6. Without being bound by theory, occlusion of the catalytic triad of TMPRSS6 may result in steric hindrance of substrate (e.g., hemojuvelin) binding to TMPRSS6.

[0100] As used herein, a TMPRSS6 antibody has a K of less than about 25 nM (e.g., at 25° C. or at 37° C.) with human TMPRSS6 protein, as measured by surface plasmon resonance, for example, using the assay format defined in Example 3 herein. DDisclosed are antibodies and antigen-binding fragments of antibodies that bind to human TMPRSS6 at a K of less than about 25 nM, less than about 20 nM, less than about 15 nM, less than about 10 nM, less than about 9 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 900 picomolar (pM), less than about 800 pM, less than about 700 pM, less than about 600 pM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, or less than about 150 pM, as measured by surface plasmon resonance, e.g., using the assay format defined in Example 3 herein, or a substantially similar assay. D In certain embodiments, the antibody or antigen-binding fragment thereof binds to human TMPRSS6 with a K in the range of about 128 pM to about 21.2 nM. D Combine with.

[0101] Also provided herein is a method for determining whether a cynomolgus monkey (e.g., Macaca fascicularis) TMPRSS6 protein has a K of less than about 30 nM (e.g., at 25° C. or at 37° C.) as measured by surface plasmon resonance using, for example, the assay format defined in Example 3 herein. D Also disclosed are antibodies and antigen-binding fragments of antibodies that bind at In certain embodiments, the antibody or antigen-binding fragment thereof has a K of less than about 30 nM, less than about 25 nM, less than about 20 nM, less than about 15 nM, less than about 10 nM, less than about 9 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 900 picomolar (pM), less than about 800 pM, less than about 700 pM, less than about 600 pM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 150 pM, less than about 100 pM, or less than about 75 pM with cynomolgus TMPRSS6, as measured by surface plasmon resonance, e.g., using an assay format defined in Example 3 herein, or a substantially similar assay. DIn certain embodiments, the antibody or antigen-binding fragment thereof binds to monkey TMPRSS6 with a K in the range of about 65 pM to about 25.7 nM. D Combine with.

[0102] Also provided herein is a method for determining whether a TMPRSS6 antibody has a K of less than about 750 nM (e.g., at 25° C. or at 37° C.) with mouse TMPRSS6 protein, as measured by surface plasmon resonance, for example, using the assay format defined in Example 3 herein. D Also disclosed are antibodies and antigen-binding fragments of antibodies that bind to mouse TMPRSS6 at less than about 750 nM, less than about 700 nM, less than about 650 nM, less than about 600 nM, less than about 550 nM, less than about 500 nM, less than about 450 nM, less than about 500 nM, less than about 5 ... K of less than about 400 nM, less than about 350 nM, less than about 300 nM, less than about 250 nM, less than about 200 nM, less than about 150 nM, less than about 50 nM, less than about 1000 picomolar (pM), less than about 900 pM, less than about 800 pM, less than about 700 pM, less than about 600 pM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 150 pM, or less than about 100 pM. D In certain embodiments, the antibody or antigen-binding fragment thereof of the present disclosure binds to mouse TMPRSS6 with a K in the range of about 82.9 pM to about 703 nM. D Combine with.

[0103] Also provided herein are antibodies that have an EC50 of less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 900 pM, less than about 800 pM, or less than about 700 pM in cells expressing human TMPRSS6, as measured, for example, using the assay format described in Example 5 herein, or a substantially similar assay. 50Also disclosed are antibodies and antigen-binding fragments thereof that bind to cells expressing human TMPRSS6 with an EC of about 670 pM to about 2.7 nM. In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure bind to cells expressing human TMPRSS6 with an EC of about 670 pM to about 2.7 nM. 50 Combine with.

[0104] Also provided herein are compounds that have an EC50 of less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, or less than about 1 nM in cells expressing cynomolgus TMPRSS6, as measured, for example, using the assay format described in Example 5 herein, or a substantially similar assay. 50 Also disclosed are antibodies and antigen-binding fragments thereof that bind to cells expressing cynomolgus monkey TMPRSS6 with an EC of about 940 pM to about 3.6 nM. In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure bind to cells expressing cynomolgus monkey TMPRSS6 with an EC of about 940 pM to about 3.6 nM. 50 Combine with.

[0105] Also provided herein are antibodies that have an EC50 of less than about 40 nM, less than about 35 nM, less than about 30 nM, less than about 25 nM, less than about 20 nM, less than about 15 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, or less than about 0.9 nM in cells expressing mouse TMPRSS6, as measured, for example, using the assay format described in Example 5 herein, or a substantially similar assay. 50 Also disclosed are antibodies and antigen-binding fragments thereof that bind to cells expressing mouse TMPRSS6 with an EC of about 840 pM to about 35 nM. In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure bind to cells expressing mouse TMPRSS6 with an EC of about 840 pM to about 35 nM. 50 Combine with.

[0106] In one embodiment, disclosed herein is an isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to TMPRSS6 protein, wherein the antibody or fragment thereof exhibits one or more of the following characteristics: (a) is a fully human monoclonal antibody; (b) has a dissociation constant (K) of less than about 21.2 nM to human TMPRSS6 at 25° C. and 37° C. as measured by a surface plasmon resonance assay. D) binds to monkey TMPRSS6 with a K of less than about 25.7 nM at 25° C. and 37° C. as measured by surface plasmon resonance assays; D (d) binds to mouse TMPRSS6 with a K of less than about 703 nM at 25° C. and 37° C. as measured by surface plasmon resonance assays. D (e) binds to cells expressing human TMPRSS6 and inhibits EC 50 (f) binds to cells expressing monkey TMPRSS6 and has an EC 50 (g) binds to cells expressing mouse TMPRSS6 and has an EC 50 has a molecular weight of less than about 35 nM; (h) HCDR1, HCDR2, and HCDR3 comprising amino acid sequences selected from the HCDR1, HCDR2, and HCDR3 sequences set forth in Table 1, and / or LCDR1, LCDR2, and LCDR3 comprising amino acid sequences selected from the LCDR1, LCDR2, and LCDR3 sequences set forth in Table 1; and / or (i) an HCVR comprising an amino acid sequence selected from the HCVR sequences set forth in Table 1, and an LCVR comprising an amino acid sequence selected from the LCVR sequences set forth in Table 1.

[0107] Disclosed herein are antibodies and antigen-binding fragments thereof that bind to TMPRSS6 and result in reduced serum iron levels in a subject in need thereof, e.g., as shown herein in Examples 10, 11, and 14.

[0108] Also disclosed herein are antibodies and antigen-binding fragments thereof that bind to TMPRSS6 and result in increased serum hepcidin concentrations in a subject in need thereof, e.g., as shown herein in Examples 10, 11, and 14.

[0109] Also disclosed herein are antibodies and antigen-binding fragments thereof that bind to TMPRSS6 and provide increased levels of mature red blood cells in subjects in need of such. In certain embodiments, antibodies and antigen-binding fragments thereof that bind to TMPRSS6 provide increased levels of mature red blood cells in the spleen and / or bone marrow in subjects in need of such. Also disclosed herein are antibodies and antigen-binding fragments thereof that bind to TMPRSS6 and provide reduced levels of reticulocytes in the spleen in subjects in need of such. Without being bound by theory, such antibodies and antigen-binding fragments thereof that bind to TMPRSS6 provide improved erythropoiesis in subjects in need of such. In certain embodiments, the antibodies and antigen-binding fragments thereof of the present disclosure bind to TMPRSS6 and provide improved erythropoiesis in subjects in need of such, reducing the need for secondary erythropoiesis in the spleen, as shown, for example, in Example 12 herein.

[0110] Also disclosed herein are antibodies and antigen-binding fragments thereof that bind to TMPRSS6 and provide increased hemoglobin levels in a subject in need thereof, for example, as shown herein in Example 13.

[0111] Disclosed herein are antibodies and antigen-binding fragments thereof that bind to TMPRSS6 and result in reduced transferrin saturation levels in a subject in need thereof, e.g., as shown herein in Example 14.

[0112] Disclosed herein is an isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to TMPRSS6 protein, which antibody or antigen-binding fragment thereof exhibits one or more of the following characteristics when administered to a subject in need thereof: (a) reduces the subject's serum iron concentration, (b) increases the subject's serum hepcidin concentration, (c) increases the subject's mature red blood cell level, (d) increases the subject's mature red blood cell level in the subject's spleen and / or bone marrow, (e) increases the subject's hemoglobin concentration, or (f) reduces the subject's transferrin saturation level.

[0113] Disclosed herein is an anti-TMPRSS6 antibody or antigen-binding fragment thereof useful for reducing serum iron concentration in a subject in need thereof. Disclosed herein is an antibody or antigen-binding fragment thereof useful for increasing serum hepcidin concentration in a subject in need thereof. Disclosed herein is an antibody or antigen-binding fragment thereof useful for increasing mature red blood cell levels in a subject in need thereof. Disclosed herein is an antibody or antigen-binding fragment thereof useful for increasing mature red blood cell levels in the spleen and / or bone marrow in a subject in need thereof. Disclosed herein is an antibody or antigen-binding fragment thereof useful for increasing hemoglobin concentration in a subject in need thereof. Disclosed herein is an antibody or antigen-binding fragment thereof useful for decreasing transferrin saturation levels in a subject in need thereof.

[0114] Disclosed herein is an anti-TMPRSS6 antibody or antigen-binding fragment thereof useful for treating or preventing at least one symptom or sign associated with an increase in serum iron concentration. In certain embodiments, the antibody or antigen-binding fragment thereof is useful for treating or preventing at least one symptom or sign associated with a decrease in serum hepcidin concentration. In certain embodiments, the antibody or antigen-binding fragment thereof is useful for treating or preventing at least one symptom or sign associated with a decrease in mature red blood cell levels. In certain embodiments, the antibody or antigen-binding fragment thereof is useful for treating or preventing at least one symptom or sign associated with an increase in mature red blood cell levels in the spleen and / or bone marrow. In certain embodiments, the antibody or antigen-binding fragment thereof is useful for treating or preventing at least one symptom or sign associated with a decrease in hemoglobin concentration. In certain embodiments, the antibody or antigen-binding fragment thereof is useful for treating or preventing at least one symptom or sign associated with an increase in transferrin saturation levels.

[0115] The antibodies and antigen-binding fragments thereof can have one or more of the aforementioned characteristics, or any combination thereof. Other characteristics of the antibodies and antigen-binding fragments thereof of the present disclosure will be apparent to those of skill in the art from a review of the present disclosure, including the working examples herein.

[0116] Inhibition of TMPRSS6 Disclosed herein are antibodies and antigen-binding fragments thereof that inhibit human TMPRSS6 in the presence of protease-dependent release of cell surface hemojuvelin (HJV), e.g., antibodies and antigen-binding fragments thereof that inhibit human TMPRSS6 and have a percent inhibition of greater than about 90% (e.g., greater than about 89.2%, greater than about 89.4%, greater than about 89.6%, greater than about 89.8%, greater than about 90%, greater than about 90.2%, greater than about 90.4%, greater than about 90.6%, greater than about 90.8%) when measured, e.g., using the assay format described in Example 6 herein, or a substantially similar assay. Also provided herein are antibodies and antigen-binding fragments thereof that inhibit human TMPRSS6 in the presence of protease-dependent release of cell surface hemojuvelin (HJV), e.g., antibodies and antigen-binding fragments thereof that inhibit human TMPRSS6 and have an IC in the range of about 70 pM to about 200 pM (e.g., about 69.3 pM, about 70 pM, about 80 pM, about 90 pM, about 100 pM, about 110 pM, about 120 pM, about 130 pM, about 140 pM, about 150 pM, about 160 pM, about 170 pM, about 180 pM, about 190 pM, about 202 pM), e.g., ... 50 Also disclosed are antibodies and antigen-binding fragments thereof that exhibit the values.

[0117] Also disclosed herein are antibodies and antigen-binding fragments thereof that inhibit mouse TMPRSS6 in the presence of protease-dependent release of cell surface hemojuvelin (HJV), e.g., antibodies and antigen-binding fragments thereof that inhibit mouse TMPRSS6 and have a percent inhibition of about 42% to about 98.5% (e.g., about 41.6%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%) when measured using, e.g., the assay format described in Example 6 herein, or a substantially similar assay. Also provided herein are antibodies and antigen-binding fragments thereof that inhibit mouse TMPRSS6 in the presence of protease-dependent release of cell surface hemojuvelin (HJV), e.g., antibodies and antigen-binding fragments thereof that inhibit mouse TMPRSS6 and have an IC in the range of about 82.6 pM to about 274 pM (e.g., about 81.8 pM, about 90 pM, about 100 pM, about 110 pM, about 120 pM, about 130 pM, about 140 pM, about 150 pM, about 160 pM, about 170 pM, about 180 pM, about 190 pM, about 200 pM, about 210 pM, about 220 pM, about 230 pM, about 240 pM, about 250 pM, about 260 pM, about 276 pM), e.g., antibodies and antigen-binding fragments thereof that inhibit mouse TMPRSS6 in the presence of protease-dependent release of cell surface hemojuvelin (HJV), e.g., antibodies and antigen-binding fragments thereof that inhibit mouse TMPRSS6 and have an IC in the range of about 82.6 pM to about 274 pM (e.g., about 81.8 pM, about 90 pM, about 100 pM, about 110 pM, about 120 pM, about 130 pM, about 140 pM, about 150 pM, about 160 pM, about 170 pM, about 180 pM, about 190 pM, about 200 pM, about 210 pM, about 220 pM, about 230 pM, about 240 pM, about 250 pM, about 260 pM, about 276 pM), e. 50 Also disclosed are antibodies and antigen-binding fragments thereof that exhibit the values.

[0118] Also disclosed herein are antibodies and antigen-binding fragments thereof that inhibit human TMPRSS6 with a percent inhibition of about 65% to about 98% (e.g., about 64.5%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98.9%), e.g., when measured using the "CM bioassay" format described in Example 7 herein, or a substantially similar assay. Also disclosed herein are antibodies and antigen-binding fragments thereof that inhibit human TMPRSS6 with an IC in the range of about 710 pM to greater than about 10 nM (e.g., about 703 pM, about 750 pM, about 800 pM, about 850 pM, about 900 pM, about 950 pM, about 1 nM, about 5 nM, about 10.1 nM), e.g., when measured using the "CM bioassay" format described in Example 7 herein, or a substantially similar assay.50 Also disclosed are antibodies and antigen-binding fragments thereof that exhibit the values.

[0119] Also disclosed herein are antibodies and antigen-binding fragments thereof that inhibit monkey TMPRSS6 with a percent inhibition of about 66% to about 98% (e.g., about 65.5%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98.9%) when measured using, for example, the "CM bioassay" format described in Example 7 herein, or a substantially similar assay. Also provided herein are compounds that inhibit monkey TMPRSS6 with an IC in the range of about 740 pM to greater than about 50 nM (e.g., about 733 pM, about 750 pM, about 800 pM, about 850 pM, about 900 pM, about 950 pM, about 1 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 35 nM, about 40 nM, about 45 nM, about 50.5 nM), as measured, for example, using the "CM bioassay" format described in Example 7 herein, or a substantially similar assay. 50 Also disclosed are antibodies and antigen-binding fragments thereof that exhibit the values.

[0120] Also disclosed herein are antibodies and antigen-binding fragments thereof that inhibit human TMPRSS6 with a percent inhibition of about 86% to about 90% (e.g., about 85.2%, about 85%, about 91%) (e.g., at 25°C or at 37°C) when measured using, for example, the "HEK293 bioassay" format described in Example 7 herein, or a substantially similar assay. Also provided herein are compounds that inhibit human TMPRSS6 (e.g., at 25° C. or at 37° C.) with an IC in the range of about 360 pM to about 5.8 nM (e.g., about 357 pM, about 400 pM, about 500 pM, about 600 pM, about 700 pM, about 800 pM, about 900 pM, about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM), as measured, for example, using the “HEK293 bioassay” format described in Example 7 herein, or a substantially similar assay. 50 Also disclosed are antibodies and antigen-binding fragments thereof that exhibit the values.

[0121] Also disclosed herein are antibodies and antigen-binding fragments thereof that inhibit monkey TMPRSS6 with a percent inhibition of about 81% to about 94% (e.g., about 80.2%, about 85%, about 90%, about 94.9%) (e.g., at 25°C or at 37°C) when measured using, for example, the "HEK293 bioassay" format described in Example 7 herein, or a substantially similar assay. Also provided herein are compounds that inhibit monkey TMPRSS6 (e.g., at 25° C. or at 37° C.) with an IC in the range of about 660 pM to about 30 nM (e.g., about 654 pM, about 700 pM, about 750 pM, about 800 pM, about 850 pM, about 900 pM, about 950 pM, about 1 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30.3 nM), as measured, for example, using the “HEK293 bioassay” format described in Example 7 herein, or a substantially similar assay. 50 Also disclosed are antibodies and antigen-binding fragments thereof that exhibit the values.

[0122] Also disclosed herein are antibodies and antigen-binding fragments thereof that inhibit mouse TMPRSS6 with a percent inhibition of about 58% to about 103% (e.g., about 57.5%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 104%) (e.g., at 25°C or at 37°C) when measured using, for example, the "HEK293 bioassay" format described in Example 7 herein, or a substantially similar assay. Also provided herein are compounds that inhibit mouse TMPRSS6 (e.g., at 25° C. or at 37° C.) with an IC in the range of about 1.5 nM to about 35 nM (e.g., about 2 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 35.3 nM), as measured, for example, using the “HEK293 bioassay” format described in Example 7 herein, or a substantially similar assay. 50 Also disclosed are antibodies and antigen-binding fragments thereof that exhibit the values.

[0123] In one embodiment, disclosed herein is an isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to TMPRSS6 protein, where the antibody or fragment thereof exhibits one or more of the following characteristics: (a) inhibits protease-dependent release of cell surface hemojuvelin in the presence of human TMPRSS6 with a percent inhibition of greater than about 90%, e.g., inhibits human TMPRSS6; (b) inhibits protease-dependent release of cell surface hemojuvelin in the presence of human TMPRSS6, e.g., inhibits human TMPRSS6 with an IC of less than about 200 pM. 50 (c) inhibits protease-dependent release of cell surface hemojuvelin in the presence of mouse TMPRSS6 with a percent inhibition of greater than about 42%, e.g., inhibits mouse TMPRSS6; (d) inhibits protease-dependent release of cell surface hemojuvelin in the presence of mouse TMPRSS6 with an IC of less than about 274 pM, e.g., inhibits mouse TMPRSS6. 50 (e) inhibits human TMPRSS6 with a percent inhibition of greater than about 65% at 25° C. and 37° C.; (f) inhibits human TMPRSS6 with an IC of less than about 10 nM at 25° C. and 37° C. 50 (g) inhibits monkey TMPRSS6 with a percent inhibition of greater than about 66% at 25° C. and 37° C.; (h) inhibits monkey TMPRSS6 with an IC of less than about 50 nM at 25° C. and 37° C. 50 (i) inhibits mouse TMPRSS6 with a percent inhibition of greater than about 58% at 25° C. and 37° C.; (j) inhibits mouse TMPRSS6 with an IC of less than about 35 nM at 25° C. and 37° C. 50(k) a fully human monoclonal antibody or an antigen-binding fragment thereof; (l) HCDR1, HCDR2, and HCDR3 having amino acid sequences selected from the HCDR1, HCDR2, and HCDR3 sequences listed in Table 1, and / or LCDR1, LCDR2, and LCDR3 having amino acid sequences selected from the LCDR1, LCDR2, and LCDR3 sequences listed in Table 1; and / or (m) an HCVR having an amino acid sequence selected from the HCVR sequences listed in Table 1, and an LCVR having an amino acid sequence selected from the LCVR sequences listed in Table 1.

[0124] The antibodies and antigen-binding fragments thereof can have one or more of the aforementioned characteristics, or any combination thereof. Other characteristics of the antibodies and antigen-binding fragments thereof of the present disclosure will be apparent to those of skill in the art from a review of the present disclosure, including the working examples herein.

[0125] Epitope mapping and related techniques Disclosed herein is an anti-TMPRSS6 antibody that interacts with one or more amino acids found in one or more regions of the TMPRSS6 protein molecule. The epitope that the antibody binds to can consist of a single continuous sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids located within any of the aforementioned domains of the TMPRSS6 protein molecule (e.g., a linear epitope within a domain). Alternatively, the epitope can consist of multiple non-contiguous amino acids (or amino acid sequences) located within any or both of the aforementioned domains of the protein molecule. In one embodiment, the epitope is a conformational epitope.

[0126] Various techniques known to those skilled in the art can be used to determine whether an antibody "interacts with one or more amino acids" in a polypeptide or protein. Exemplary techniques include routine cross-blocking assays, such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY). Other techniques include alanine scanning mutation analysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248:443-63), peptide truncation analysis, crystal structure studies, and NMR analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be used (Tomer (2000) Prot. Sci. 9:487-496).

[0127] Another method that can be used to identify amino acids in a polypeptide with which an antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. See, for example, Example 8 of the present disclosure. Broadly speaking, the hydrogen / deuterium exchange method involves labeling a protein of interest with deuterium and then binding an antibody to the deuterium-labeled protein. When the protein / antibody complex is then transferred to water, exchangeable protons in amino acids protected by the antibody complex undergo back exchange from deuterium to hydrogen at a slower rate than exchangeable protons in amino acids that are not part of the interface. As a result, amino acids that form part of the protein / antibody interface may retain deuterium and therefore exhibit a relatively higher mass compared to amino acids that are not included in the interface. After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry, thereby revealing deuterium-labeled residues that correspond to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267:252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0128] Modification-Assisted Profiling (MAP), also known as Antigen Structure-based Antibody Profiling (ASAP), is a method to classify multiple monoclonal antibodies (mAbs) against the same antigen according to the similarity of their binding profile to chemically or enzymatically modified antigen surfaces (see US2004 / 0101920, expressly incorporated herein by reference in its entirety). Each category may reflect a unique epitope that is either distinct or overlapping with the epitope represented by another category. This technique allows for rapid filtering of genetically identical antibodies, and characterization can focus on genetically distinct antibodies. When applied to hybridoma screening, MAP can facilitate the identification of rare hybridoma clones that produce mAbs with desired properties. MAP can be used to classify the antibodies disclosed herein into groups of antibodies that bind to different epitopes.

[0129] The present disclosure includes anti-TMPRSS6 antibodies that bind to the same epitope or portion of an epitope as any of the specific exemplary antibodies set forth in Table 1. Also disclosed herein are anti-TMPRSS6 antibodies that compete with any of the specific exemplary antibodies set forth in Table 1 for binding to a TMPRSS6 protein or fragment thereof. For example, the present disclosure includes anti-TMPRSS6 antibodies that cross-compete with one or more antibodies set forth in Table 1 for binding to a TMPRSS6 protein.

[0130] Whether an antibody binds to the same epitope as a reference anti-TMPRSS6 antibody or competes for binding can be determined using routine methods known in the art.For example, to determine whether a test antibody binds to the same epitope as a reference anti-TMPRSS6 antibody disclosed herein, the reference antibody is bound to TMPRSS6 protein or peptide under saturation conditions.Then, the ability of the test antibody to bind to TMPRSS6 protein molecule is evaluated.If the test antibody can bind to TMPRSS6 after saturation binding with the reference anti-TMPRSS6 antibody, it can be concluded that the test antibody binds to an epitope different from the epitope of the reference anti-TMPRSS6 antibody.On the other hand, if the test antibody cannot bind to TMPRSS6 protein after saturation binding with the reference anti-TMPRSS6 antibody, the test antibody can bind to the same epitope as the epitope bound by the reference anti-TMPRSS6 antibody disclosed herein.

[0131] To determine whether an antibody competes with a reference anti-TMPRSS6 antibody for binding, the above binding method is carried out in two directions: in the first direction, the reference antibody is allowed to bind to TMPRSS6 protein under saturating conditions, and then the binding of the test antibody to the TMPRSS6 molecule is evaluated. In the second direction, the test antibody is allowed to bind to the TMPRSS6 molecule under saturating conditions, and then the binding of the reference antibody to the TMPRSS6 molecule is evaluated. If only the first (saturating) antibody can bind to the TMPRSS6 molecule in either direction, it is concluded that the test antibody and the reference antibody compete for binding to TMPRSS6. As will be understood by those skilled in the art, an antibody that competes with the reference antibody for binding does not necessarily bind to the same epitope as the reference antibody, but may sterically block the binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0132] Two antibodies bind to the same or overlapping epitopes if each of them competitively inhibits (blocks) the binding of the other to the antigen. That is, 1-fold, 5-fold, 10-fold, 20-fold or 100-fold excess of one antibody inhibits the binding of the other by at least 50%, preferably 75%, 90% or even 99%, as measured by competitive binding assay (see, for example, Junghans et al., Cancer Res. 1990 50:1495-1502). Alternatively, two antibodies have the same epitope if essentially all of the amino acid mutations of the antigen that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other. Two antibodies have overlapping epitopes if some of the amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other.

[0133] Additional routine experiments (e.g., peptide mutations and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antibody is indeed due to binding to the same epitope as the reference antibody, or whether steric blocking (or another phenomenon) is responsible for the observed lack of binding. This type of experiment can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0134] immune complex The present disclosure encompasses human anti-TMPRSS6 monoclonal antibodies conjugated to a therapeutic moiety ("immunoconjugate") for treating diseases or disorders associated with TMPRSS6 (e.g., iron overload disorders). As used herein, the term "immunoconjugate" refers to an antibody chemically or biologically linked to a radioactive substance, cytokine, interferon, target or reporter moiety, enzyme, peptide or protein, or therapeutic agent. The antibody may be linked to a radioactive substance, cytokine, interferon, target or reporter moiety, enzyme, peptide, or therapeutic agent at any position along the molecule, so long as it is capable of binding to its target. Examples of immunoconjugates include antibody drug conjugates and antibody-toxin fusion proteins. In one embodiment, the drug may be a second, different antibody against the TMPRSS6 protein. The type of therapeutic moiety that may be conjugated to the anti-TMPRSS6 antibody takes into consideration the condition to be treated and the desired therapeutic effect to be achieved. Examples of suitable materials for forming immunoconjugates are known in the art, see, for example, WO05 / 103081.

[0135] Therapeutic Administration and Formulations The present disclosure provides therapeutic compositions comprising the anti-TMPRSS6 antibody or antigen-binding fragment thereof disclosed herein. The therapeutic compositions according to the present disclosure are administered with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved incorporation, delivery, tolerability, and the like. Many suitable formulations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, a formulary known to every pharmacist. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0136] A variety of delivery systems are known and can be used to administer the pharmaceutical compositions disclosed herein, such as, for example, liposomal encapsulation, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, for example, Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral, rectal, and intestinal mucosa, etc.), and can be administered together with other biologically active agents. Administration can be systemic or local. Pharmaceutical compositions can also be delivered in vesicles, in particular liposomes (see, eg, Langer (1990) Science 249:1527-1533).

[0137] The use of nanoparticles to deliver the antibodies disclosed herein is also contemplated herein. Antibody-conjugated nanoparticles can be used for both therapeutic and diagnostic applications. Antibody-conjugated nanoparticles and methods of preparation and use are detailed by Arruebo, M., et al. 2009 ("Antibody-conjugated nanoparticles for biomedical applications" in J.Nanomat.Volume 2009, Article ID 439389, page 24, doi:10.1155 / 2009 / 439389), which are incorporated herein by reference. Nanoparticles may be made to be conjugated to antibodies contained in pharmaceutical compositions that target cells. Nanoparticles for drug delivery are also described, for example, in US8257740 or US8246995, each of which is incorporated herein in its entirety.

[0138] In certain circumstances, the pharmaceutical composition may be delivered in a controlled release system. In one embodiment, a pump may be used. In another embodiment, a polymeric material may be used. In yet another embodiment, the controlled release system may be placed in close proximity to the target of the composition, thus requiring only a small systemic dose.

[0139] The preparations for injection may include dosage forms for intravenous, subcutaneous, intracranial, intraperitoneal and intramuscular injection, drip infusion, etc. These preparations for injection may be prepared by known methods.

[0140] The pharmaceutical composition of the present disclosure can be delivered subcutaneously or intravenously using a standard needle and syringe. Furthermore, for subcutaneous delivery, a pen delivery device is easily applied in delivering the pharmaceutical composition as disclosed herein. Such a pen delivery device can be either reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all the pharmaceutical composition in the cartridge is administered, the cartridge is emptied, and the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be used again. Disposable pen delivery devices do not have a replaceable cartridge. Rather, disposable pen delivery devices are pre-filled with the pharmaceutical composition and held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0141] Advantageously, the pharmaceutical compositions for oral or parenteral use are prepared in dosage forms with unit doses suited to the dosage of the active ingredient, such as tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0142] Therapeutic Uses of Antibodies The anti-TMPRSS6 antibodies and antigen-binding fragments thereof of the present disclosure are useful for treating and / or preventing a disease or disorder or condition associated with TMPRSS6, and / or for ameliorating at least one symptom associated with such a disease, disorder or condition. In certain embodiments, the anti-TMPRSS6 antibodies or antigen-binding fragments thereof of the present disclosure may be administered in therapeutic doses to a subject having a disease or disorder or condition associated with TMPRSS6.

[0143] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure are useful for treating or preventing at least one symptom or sign of a disease or disorder associated with TMPRSS6. In certain embodiments, the disease or disorder associated with TMPRSS6 is an iron overload disorder. In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure are useful for treating or preventing at least one symptom of congenital dyserythroplastic anemia, Diamond-Blackfan anemia, alpha thalassemia, beta thalassemia, transfusion-dependent hemolytic anemia, myelodysplastic syndrome, sickle cell disease, polycythemia vera, hereditary hemochromatosis, primary hemochromatosis, secondary hemochromatosis, severe juvenile hemochromatosis, or chronic liver disease.

[0144] In certain embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is useful for treating or preventing at least one symptom, complication, or sign of beta thalassemia. In certain embodiments, the beta thalassemia is beta thalassemia major or beta thalassemia intermediate. Symptoms of beta thalassemia include fatigue, weakness, pale or yellowish skin, facial bone deformity, slow growth, abdominal distension, dark urine, and other complications (e.g., complications from treatment). Examples of complications of moderate to severe beta thalassemia include iron overload, which can be the result of either disease or frequent blood transfusions, and is associated with damage to organs such as the heart, liver, and endocrine system (e.g., hormone-producing glands that regulate processes throughout the body), and increased risk of infection. Complications of severe beta thalassemia can also include bone deformity, enlarged spleen, slow growth rate, and heart problems.

[0145] In certain embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is useful for treating or preventing at least one symptom, complication, or sign of hemochromatosis.Hemochromatosis symptoms include, for example, joint pain, abdominal pain, weakness, fatigue, diabetes, sexual desire, impotence, heart failure, liver failure, bronze or gray skin color, and memory blur.Hemochromatosis complications include, for example, liver problems, pancreatic problems, heart problems, reproductive problems, and skin color changes.

[0146] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure are useful for treating or preventing at least one symptom, complication, or sign associated with iron overload. Iron overload can be the result of an inherited disease or disorder, or the result of receiving frequent blood transfusions, consuming high concentrations of iron supplements, and / or receiving injections containing iron. Genetic diseases that result in iron overload include various anemias such as hereditary hemochromatosis, sickle cell disease, and sideroblastic anemia, African iron overload (e.g., when the affected person drinks beverages that contain a lot of iron), enzyme deficiencies (e.g., pyruvate kinase deficiency, glucose-6-phosphate dehydrogenase deficiency), and protein transport disorders such as aceruloplasminemia and atransferrinemia.

[0147] Symptoms of iron overload include, for example, fatigue (e.g., chronic fatigue), joint pain, abdominal pain, liver disease (e.g., cirrhosis, liver cancer, diabetes mellitus, heart problems (e.g., irregular heart rhythm, heart attack, heart failure), changes in skin color (e.g., bronze, gray-green), loss of period, loss of interest in sex, impotence, infertility, hypogonadism, bone problems (e.g., osteoarthritis, osteoporosis), hair loss, enlarged liver or spleen, hypothyroidism, hypopituitarism, depression, adrenal problems, early onset neurodegenerative diseases (e.g., Alzheimer's disease, early onset Parkinson's disease, Huntington's disease, epilepsy, multiple sclerosis), and elevated markers such as blood glucose, liver enzymes, serum iron, serum transferrin, and serum ferritin.

[0148] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure are useful for treating or preventing at least one symptom or sign associated with receiving a blood transfusion, e.g., receiving frequent and / or regular blood transfusions. In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure are useful for treating or preventing at least one symptom or sign associated with a physical trauma resulting in blood loss. In certain embodiments, the physical trauma resulting in blood loss requires treatment with a blood transfusion. Thus, the symptom or sign associated with a physical trauma resulting in blood loss can be any symptom or sign associated with treatment of a physical trauma with a blood transfusion (e.g., iron overload). For example, the physical trauma can be a traumatic accident resulting in blood loss (e.g., loss of limb, blunt force trauma, leading to deep wounds) requiring a blood transfusion. In certain embodiments, the transfusion(s) required is a large volume of blood transfusion.

[0149] In certain embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is useful for treating or preventing at least one symptom or sign of myelodysplastic syndrome. In certain embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is useful for treating or preventing at least one symptom or sign of myelodysplastic syndrome with ringed sideroblasts. In certain embodiments, the disclosed antibody or antigen-binding fragment thereof of the present disclosure is useful for treating or preventing at least one symptom or sign of transfusion-dependent hemolytic anemia. In certain embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is useful for treating or preventing at least one symptom or sign of transfusion-dependent hemolytic anemia due to pyruvate kinase deficiency (i.e., hemolytic anemia) or sideroblastic transfusion-dependent hemolytic anemia (i.e., sideroblastic anemia). In certain embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is useful for treating or preventing at least one symptom or sign of sickle cell anemia. In certain embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is useful for treating or preventing at least one symptom or sign of dyserythroid anemia. In certain embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is useful for treating or preventing at least one symptom or sign of erythropoietic porphyria. In certain embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is useful for treating or preventing at least one symptom or sign of chronic liver disease. In certain embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is useful for treating or preventing at least one symptom or sign of alcohol-related chronic liver disease, hepatitis C, or autoimmune hepatitis.

[0150] Also disclosed is the prophylactic use of an antibody or antigen-binding fragment thereof of the present disclosure in a subject at risk of suffering from a disease or disorder associated with TMPRSS6.

[0151] In some embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure are used for the preparation of a pharmaceutical composition or medicament for treating a subject suffering from a disease, disorder, or condition disclosed herein. In another embodiment, the antibodies or antigen-binding fragments thereof of the present disclosure are used as an adjunct therapy with any other agent or any other therapy known to one of skill in the art useful for treating or ameliorating a disease, disorder, or condition disclosed herein.

[0152] Combination therapy The combination therapy may include one or more of the antibodies or antigen-binding fragments thereof of the present disclosure and any additional therapeutic agent that can be advantageously combined with the antibodies or antigen-binding fragments thereof of the present disclosure. The antibodies or antigen-binding fragments of the present disclosure may be synergistically combined with one or more drugs or therapies used to treat diseases or disorders associated with TMPRSS6. In some embodiments, the antibodies or antigen-binding fragments of the present disclosure may be combined with a second therapeutic agent to improve one or more symptoms of the disease or condition.

[0153] Depending on the disease, disorder, or condition, the antibodies or antigen-binding fragments disclosed herein may be used in combination with one or more additional therapeutic agents. For example, if the disease, disorder, or condition is associated with iron overload, the antibodies or antigen-binding fragments disclosed herein may be used in combination with one or more iron depletion therapies and / or therapies currently used to treat iron overload.

[0154] In certain embodiments, iron chelation therapy may be used to treat iron overload. Iron chelation therapy is the pharmacological removal of iron using an iron chelator. Examples of iron chelators include deferoxamine, deferasirox, and deferiprone. (See, e.g., Mobarra et al., Int. J. Hematol. Oncol. Stem Cell Res. (2016) 10(4): 239-247). Other examples of iron chelators are known to those skilled in the art, such as those reviewed in Hatcher et al., Future Med. Chem. (2009) 1(9): 1643-70.

[0155] Other therapies that may be used to treat iron overload include, without limitation, the use of drugs such as vitamin E, wheat germ oil, tocophersolan, indicaxanthin, folic acid, drugs that increase fetal hemoglobin concentrations (e.g., hydroxyurea, histone deacetylase inhibitors and / or DNA methyltransferase inhibitors (e.g., butyric acid derivatives, azacitidine, decitabine (5-aza 2-deoxycytidine) and trichostatin-A), hemin, pomalidomide, thalidomide, cytokines such as stem cell factor (SCF) and transforming growth factor beta (TGF beta)), drugs to manage ulcers, drugs to manage infections (e.g., antibiotics and antivirals), drugs to treat thrombosis, or drugs to treat anemia (e.g., luspatercept).

[0156] Iron overload can also be treated with stem cell or bone marrow transplants, blood transfusions, or bloodletting.

[0157] As used herein, the term "in combination with" means that the additional therapeutically active ingredient(s) may be administered prior to, simultaneously with, or following administration of an anti-TMPRSS6 antibody or antigen-binding fragment disclosed herein. The term "in combination with" also includes sequential or concomitant administration of one or more of the anti-TMPRSS6 antibodies or antigen-binding fragments of the present disclosure and a second therapeutic agent.

[0158] The additional therapeutically active ingredient(s) may be administered to a subject prior to administration of one or more of the anti-TMPRSS6 antibodies or antigen-binding fragments thereof of the present disclosure. For example, if the first component is administered 1 week, 72 hours, 60 hours, 48 ​​hours, 36 hours, 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, or more than 30 minutes before administration of the second component, the first component may be considered to be administered "before" the second component.

[0159] In other embodiments, the additional therapeutically active component(s) may be administered to the subject after administration of one or more of the anti-TMPRSS6 antibodies or antigen-binding fragments thereof of the present disclosure. For example, if the first component is administered 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, or more after administration of the second component, the first component may be considered to be administered "after" the second component.

[0160] In other embodiments, the additional therapeutically active ingredient(s) may be administered to a subject simultaneously with administration of one or more of the anti-TMPRSS6 antibodies or antigen-binding fragments thereof of the present disclosure. "Concurrent" administration, for purposes of this disclosure, includes, for example, administration of one or more of the anti-TMPRSS6 antibodies or antigen-binding fragments thereof and the additional therapeutically active ingredient to a subject in a single dosage form or in separate dosage forms administered to a subject within about 30 minutes of each other. When administered in separate dosage forms, each dosage form may be administered by the same route (e.g., both the anti-TMPRSS6 antibody and the additional therapeutically active ingredient may be administered intravenously, etc.), or alternatively, each dosage form may be administered by a different route (e.g., the disclosed antibody or antigen-binding fragment may be administered intravenously, and the additional therapeutically active ingredient may be administered orally). Administration of the ingredients in a single dosage form, separate dosage forms by the same route, or separate dosage forms by different routes, are all considered "concurrent administration" for purposes of this disclosure. For purposes of this disclosure, administration of one or more of the anti-TMPRSS6 antibodies or antigen-binding fragments thereof of the present disclosure "before," "concurrently with," or "after" the administration of an additional therapeutically active ingredient (as those terms are defined herein above) is considered administration of the anti-TMPRSS6 antibodies or antigen-binding fragments thereof of the present disclosure "in combination with" the additional therapeutically active ingredient.

[0161] Also disclosed herein are pharmaceutical compositions in which the anti-TMPRSS6 antibodies or antigen-binding fragments disclosed herein are combined with one or more additional therapeutically active ingredient(s) described elsewhere herein.

[0162] Diagnostic Uses of Antibodies The antibodies and antigen-binding fragments thereof of the present disclosure may be used to detect and / or measure TMPRSS6 in a sample, for example, for diagnostic purposes. Disclosed herein is the use of the antibodies and antigen-binding fragments thereof in an assay for detecting a disease or disorder associated with TMPRSS6. An exemplary diagnostic assay for TMPRSS6 may include, for example, contacting a sample obtained from a patient with an anti-TMPRSS6 antibody or antigen-binding fragment thereof disclosed herein that is labeled with a detectable label or reporter molecule. The antibodies and antigen-binding fragments thereof of the present disclosure may also be used as a capture ligand to selectively isolate TMPRSS6 from a sample from a subject. Alternatively, an unlabeled anti-TMPRSS6 antibody or fragment may be used for diagnostic purposes in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule may be a 3 H, 14 C. 32 P, 35 S, or 125 The TMPRSS6 signaling molecule may be a radioisotope such as I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Certain exemplary assays that can be used to detect or measure TMPRSS6 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0163] The sample that can be used for TMPRSS6 diagnostic assay includes any tissue sample or body fluid sample that can be obtained from a patient and contains detectable amount of TMPRSS6 protein or its fragment under normal or pathological condition.Generally, the concentration of TMPRSS6 protein in a particular sample obtained from a healthy subject (e.g., a patient who does not suffer from a disease associated with TMPRSS6) can be measured to first establish the baseline or standard concentration of TMPRSS6.Then, this baseline concentration of TMPRSS6 can be compared with the concentration of TMPRSS6 measured in a sample obtained from an individual suspected of having a TMPRSS6-related condition or a symptom associated with such a condition.

[0164] Anti-TMPRSS6 antibodies or antigen-binding fragments thereof may contain no additional label or moiety, or they may contain N-terminal or C-terminal label or moiety. In one embodiment, the label or moiety is biotin. In binding assays, the position of the label (if present) can determine the orientation of the peptide relative to the surface to which it is bound. For example, if the surface is coated with avidin, the peptide containing N-terminal biotin is oriented such that the C-terminus of the peptide is distal to the surface. EXAMPLES

[0165] The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions disclosed herein, and are not intended to limit the scope of what the inventors regard as their invention. Attempts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, room temperature is about 25° C., and pressure is at or near atmospheric pressure.

[0166] Example 1: Generation of human antibodies against transmembrane serine protease 6 (TMPRSS6) Human antibodies against the TMPRSS6 protein were generated in VELOCIMMUNE® mice, which contain DNA encoding the variable regions of human immunoglobulin heavy and kappa light chains. Mice were immunized and boosted with human TMPRSS6 DNA by hydrodynamic DNA delivery.

[0167] Anti-TMPRSS6 antibodies were isolated directly from B cells of antigen-positive mice without fusion to myeloma cells, as described in U.S. Patent No. 7,582,298, the entire contents of which are expressly incorporated herein by reference. Using this method, several fully human anti-TMPRSS6 antibodies (i.e., antibodies with human variable and human constant domains) were obtained.

[0168] Exemplary antibodies generated as disclosed above are designated mAb37746, mAb37763, mAb37777, mAb37699, mAb41422, mAb41465, and mAb41450, and have the amino acid and nucleic acid sequences of HCVR, HCDR1, HCDR2, HCDR3, LCVR, LCDR1, LCDR2, and LCDR3 identified in Tables 1 and 2.

[0169] The biological properties of exemplary antibodies generated according to the methods of this example are detailed in the Examples below.

[0170] Example 2: Amino acid and nucleotide sequences of the heavy and light chain variable regions Table 1 shows the heavy and light chain variable region sequences, the CDR sequences, and amino acid sequence identifiers for the heavy and light chain sequences of selected exemplary anti-TMPRSS6 antibodies. [Table 1]

[0171] The corresponding nucleic acid sequence identifiers for selected exemplary anti-TMPRSS6 antibodies are set forth in Table 2. [Table 2]

[0172] The antibodies may have a human or mouse Fc isotype. As will be appreciated by those of skill in the art, an antibody having a particular Fc isotype can be converted to an antibody having a different Fc isotype (e.g., an antibody having a mouse IgG1 Fc can be converted to an antibody having a human IgG1 or human IgG4 Fc, etc.), but in each case the variable domains (including the CDRs) indicated by the numerical identifiers in Table 1 remain the same, and the binding characteristics to the antigen are expected to be the same or substantially similar regardless of the nature of the Fc domain.

[0173] Unless otherwise indicated, all antibodies used in the following examples contain human IgG4 Fc with serine to proline mutation in the hinge region (S108P). Exemplary antibodies containing human IgG4 Fc with serine to proline mutation in the hinge region (S108P) are named REGN7969, REGN7971, REGN7973, REGN8023, REGN7974, REGN7977, and REGN7999, respectively: mAb37746, mAb37763, mAb37777, mAb37699, mAb41422, mAb41465, and mAb41450. Table 3 shows the amino acid sequence identifiers of the full-length heavy chain sequence and full-length light chain sequence of these antibodies. [Table 3]

[0174] Example 3: Antibodies that bind to TMPRSS6 as determined by surface plasmon resonance Experimental procedure The equilibrium dissociation constants (K D) was determined using a real-time surface plasmon resonance biosensor using a Biacore 4000 instrument. All binding studies were performed at 25° C. and 37° C. in a running buffer of 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05 v / v% Surfactant Tween-20, pH 7.4 (HBS-ET). To capture the different TMPRSS6 mAbs, the surface of a Biacore CM5 sensor chip was first derivatized by amine coupling with a human Fc-specific mouse mAb. Several TMPRSS6 reagent proteins were used in the experiments including human TMPRSS6 extracellular domain with an S762A mutation expressed with a C-terminal myc-myc-hexahistidine tag (hTMPRSS6_S762A-MMH; referred to as REGN5330; SEQ ID NO: 117), Macaca fascicularis TMPRSS6 extracellular domain with an S751A mutation expressed with a C-terminal myc-myc-hexahistidine tag (mfTMPRSS6_S751A-MMH; referred to as REGN5977; SEQ ID NO: 119), and mouse TMPRSS6 extracellular domain with an S762A mutation expressed with a C-terminal myc-myc-hexahistidine tag (mTMPRSS6_S762A-MMH; referred to as REGN6848; SEQ ID NO: 118). Different concentrations of TMPRSS6 reagent protein (100 nM-3.7 nM, 3-fold serial dilutions) prepared in HBS-EP running buffer were injected over the TMPRSS6 mAb-captured surface at a flow rate of 30 μL / min for 150 s, and their dissociation in HBS-ET running buffer was monitored for 10 min. At the end of each cycle, the TMPRSS6 mAb-captured surface was regenerated with a 12 s injection of 20 mM phosphoric acid.

[0175] Association rates (k ) were calculated by fitting the real-time binding sensorgrams to a 1:1 binding model with mass transport limitation using Scrubber 2.0 (BioLogic Software) curve fitting software. a ) and dissociation rate (k d The binding / dissociation equilibrium constant (KD ) and dissociation half-life (t1 / 2),

number

[0176] result The binding kinetic parameters of the different selected TMPRSS6 mAbs to each TMPRSS6 reagent protein are shown in Tables 4 to 9. [Table 4]

[0177] As shown in Table 4, the selected TMPRSS6 mAbs had K values ​​ranging from about 128 pM to about 2.63 nM for hTMPRSS6_S762A-MMH at 25°C. D Combined by value. [Table 5]

[0178] As shown in Table 5, the selected TMPRSS6 mAbs had K values ​​ranging from about 2.24 nM to about 21.2 nM for hTMPRSS6_S762A-MMH at 37°C. D Combined by value. [Table 6]

[0179] As shown in Table 6, the selected TMPRSS6 mAbs had K values ​​ranging from about 65 pM to about 7.18 nM for mfTMPRSS6_S751A-MMH at 25°C. D Combined by value. [Table 7]

[0180] As shown in Table 7, the selected TMPRSS6 mAbs had K values ​​ranging from about 750 pM to about 25.7 nM for mfTMPRSS6_S751A-MMH at 37°C. D Combined by value. [Table 8]

[0181] As shown in Table 8, three of the seven selected TMPRSS6 mAbs showed K values ​​ranging from about 82.9 pM to about 233 nM for mTMPRSS6_S762A-MMH at 25 °C. D Combined by value. [Table 9]

[0182] As shown in Table 9, three of the seven selected TMPRSS6 mAbs showed K values ​​ranging from about 317 pM to about 703 nM for mTMPRSS6_S762A-MMH at 37 °C. D Combined by value.

[0183] Example 4: Cross-competition between selected anti-TMPRSS6 monoclonal antibodies Experimental procedure Binding competition between TMPRSS6 monoclonal antibodies (mAbs) was determined using a real-time label-free biolayer interferometry (BLI) assay on an Octet HTX biosensor platform (Pall ForteBio Corp.). The entire experiment was carried out at 25°C in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% v / v Surfactant Tween-20, 1 mg / mL BSA, 0.02% NaN3, pH 7.4 (HBS-EBT) buffer with plates shaking at a speed of 1000 rpm. To assess whether the two mAbs can compete with each other for binding to their respective epitopes on TMPRSS6, a recombinant protein consisting of the extracellular domain of human TMPRSS6 expressed with the S762A mutation and a C-terminal myc-myc-hexahistidine tag (hTMPRSS6_S762A-MMH; designated REGN5330, SEQ ID NO: 117) was first captured on a biosensor chip by immersing an Octet biosensor chip coated with an anti-penta-His antibody (HIS1K) in a well containing a solution of 10 μg / mL of hTMPRSS6_S762A-MMH for 1 minute. The biosensor chip with captured hTMPRSS6_S762A-MMH was then saturated with the first TMPRSS6 mAb (designated mAb-1) by immersing the chip in a well containing a solution of 50 μg / mL of mAb-1 for 4 minutes. The biosensor chip was then subsequently immersed in a well containing a solution of 50 μg / mL of the second TMPRSS6 mAb (referred to as mAb-2) for 3 minutes. Between each step of the experiment, the biosensor chip was washed in HBS-EBT buffer. The real-time binding response was monitored throughout the course of the experiment, and the binding response at the end of each step was recorded.

[0184] result The responses of mAb-1 and mAb-2 binding to pre-complexed hTMPRSS6_S762A-MMH were compared and the competitive / non-competitive behavior of the different selected TMPRSS6 mAbs was determined as shown in Table 10. [Table 10]

[0185] Example 5: Antibodies that bind to cells expressing TMPRSS6 Experimental procedure To evaluate cell binding by the anti-TMPRSS6 antibodies of the present disclosure, HEK293 cells were engineered to stably overexpress full-length TMPRSS6 from human (NCBI Reference Sequence NM_001289001), cynomolgus monkey / Macaca fascicularis (NCBI Reference Sequence XM_005567384), and mouse (NCBI Reference Sequence NM_027902.2), and these cell lines are referred to as HEK293 / hTMPRSS6, HEK293 / mfTMPRSS6, and HEK293 / mTMPRSS6, respectively. Additionally, HEK293 cells were engineered to stably overexpress catalytically inactive full-length human TMPRSS6, and the serine at position 753 of NCBI Reference Sequence NM_001289001 was changed to alanine. This cell line is referred to as HEK293 / hTMPRSS6(S762A). None of the cell lines were sorted, except for the mouse TMPRSS6 cell line (HEK293 / mTMPRSS6), which was sorted for high expression of mouse TMPRSS6.

[0186] To assess binding of the anti-TMPRSS6 antibodies of the present disclosure to the receptor expressed on the cell surface, the antibodies were incubated in an assay buffer (Ca ++ and Mg ++ Antibodies (including IgG subclass controls) were serially diluted from 375 nM to 22.9 pM in PBS containing 2% FBS without IgG (plus sample-containing assay buffer alone without the test molecule) and diluted at 0.5 × 10 in assay buffer. 6Cells / well were incubated for 30 minutes at 4°C. After incubation with primary antibody, cells were stained with 4.0 μg / mL Alexa Fluor®-647-conjugated secondary antibody (Jackson ImmunoResearch Laboratories Inc., anti-human #109-607-003) for 30 minutes at 4°C. Cells were fixed using BD CytoFix™ (Becton Dickinson, #554655) and analyzed on either an IQue® or IQue® Plus Flow Cytometer (Intellicyt®). Unstained and secondary antibody alone controls were also run for all cell lines. Results were analyzed using ForeCyt® (IntelliCyt®) software to determine the geometric mean of fluorescence of viable cells and calculate percent binding by normalizing the geometric mean of the test conditions with the geometric mean of the corresponding unstained cells.

[0187] result Table 11 shows the binding of selected anti-TMPRSS6 antibodies to cells engineered to express hTMPRSS6, hTMPRSS6(S762A), mfTMPRSS6, and mTMPRSS6. [Table 11]

[0188] As shown in Table 11, the selected anti-TMPRSS6 antibodies showed binding to human, cynomolgus monkey, and human S762A TMPRSS6 expressed in HEK293 cells. Maximum fold binding and EC 50 The range of values ​​was about 47-69 fold and about 670 pM-2.7 nM for hTMPRSS6 expressing cells, about 145-420 fold and about 1.6-2.3 nM for hTMPRSS6(S762A) expressing cells, and about 50-91 fold and about 940 pM-3.6 nM for mfTMPRSS6 expressing cells. The maximum binding fold and EC 50The range of values ​​was about 42-421 fold and about 840 pM-35 nM. The selected anti-TMPRSS6 antibodies showed binding to parental HEK293 cells at binding rates of about 3-5 fold. The IgG subclass control antibody (hIgG subclass control) samples and secondary antibody alone (anti-human 2" alone) samples showed binding rates in the range of about 1-3 fold.

[0189] Example 6: Detection of Human Hemojuvelin (HJV) Experimental procedure To further characterize the ability of anti-TMPRSS6 monoclonal antibodies (mAbs) to inhibit TMPRSS6, a cell-based bioassay with two different readouts was used. TMPRSS6 acts as a negative regulator of hepcidin expression by cleaving cell surface hemojuvelin (HJV). See, e.g., Rausa et al. J. Cell. Mol. Med. (2015) 19:879-888, and Lee P., Acta Haematol. (2009) 122:87-96.

[0190] For the assay, flow cytometry was used to measure cell surface human hemojuvelin (hHJV) and ELISA assays were performed to quantify soluble hHJV levels in HEK293 cells expressing full-length human HJV and either human or mouse TMPRSS6. hHJV-expressing HEK293 cells (hHJV; amino acids 1-426, NCBI reference sequence NP_998818.1) were used as the parental cell line to engineer co-expression bioassay cell lines. Parental cell lines stably expressing human HJV were selected using 500 μg / mL of G418 in Dulbecco's modified Eagle's medium (DMEM) containing 10% (v / v) fetal bovine serum, 100 units of penicillin, 100 μg of streptomycin and 292 μg / ml of L-glutamine. These hHJV-expressing HEK293 cells (HEK293.hHJV) were then enriched for high cell surface expression of hHJV by FACS (anti-hemojuvelin antibody; Abcam ab54431, donkey anti-mouse IgG AF647; Invitrogen A-31571). To express TMPRSS6, HEK293.hHJV were transduced with lentiviral supernatants for either full-length human TMPRSS6 (hTMPRSS6; amino acids 1-811, NCBI reference sequence NM_153609.3) or full-length mouse TMPRSS6 (mTMPRSS6; amino acids 1-811, NCBI reference sequence NM_027902.2), containing a C-terminal triple myc tag. The resulting cell lines were designated HEK293.hHJV / 3xMyc.hTMPRSS6 and HEK293.hHJV / 3xMyc.mTMPRSS6, respectively.) Lentiviral-transduced cells were further selected with 500 μg / mL G418 and 100 μg / mL hygromycin B for 2 weeks and sorted for high-expressing TMPRSS6 populations using anti-Myc-AF647 antibody.

[0191] For ELISA, assay cell lines were plated in flat-bottom 96-well plates at 1 × 10 4Cells / well were seeded. Antibodies and controls of the present disclosure were serially diluted (from 0 nM to 200 nM) and then added to each well containing a final volume of 100 μL, followed by overnight incubation at 37° C. The following day, supernatants were collected and ELISA was performed using the Human RGM-C / Hemojuvelin DuoSet ELISA kit (R&D Systems, DY3720-05) according to the manufacturer's operating instructions. Optical density was acquired using a SpectraMax i3 (Molecular Devices), and the results were analyzed with Prism software (GraphPad) to determine IC 50 The value was obtained from the following equation:

number

[0192] In this equation, "soluble HJV ベースライン " indicates the amount of soluble HJV from the supernatant of HJV-TMPRSS6-overexpressing cells without antibody or protease inhibitor treatment. 抗体 " indicates the amount of soluble HJV from the assay sample treated with the highest concentration of antibody, and "soluble HJV プロテアーゼ阻害剤 " indicates the amount of soluble HJV from samples treated with the serine protease inhibitor aprotinin (Sigma, Cat: 10236624001).

[0193] For FACS analysis, HEK293.hHJV / 3xMyc.hTMPRSS6 or HEK293.hHJV / 3xMyc.mTMPRSS6 cells were treated with 50 nM anti-TMPRSS6 antibody overnight. The next day, cells were harvested for surface staining. To stain cell surface hHJV in the assay cell lines, cells were blocked with FcR blocker (Miltenyi Biotech, 130-059-901) and stained with anti-hemojuvelin antibody (Abcam ab54431, 1:400, on ice, 1 hour), followed by incubation with donkey anti-mouse IgG AF647 antibody (Invitrogen A-31571, 1:2000, on ice, 40 minutes). Stained cells were measured using an Accuri C6 flow cytometer (BD).

[0194] In the ELISA assay, the serine protease inhibitor aprotinin (2 μg / mL) and a protease inhibitor cocktail (Sigma, Cat: P-1860, 1:200) were included as controls to block protease activity in HEK293.hHJV / 3xMyc.TMPRSS6 cells. In the FACS, aprotinin (2 μg / mL) was included as a control to block protease activity in HEK293.hHJV / 3xMyc.TMPRSS6 cells.

[0195] result Table 12 shows the inhibition of protease-dependent release of HJV from the cell surface by anti-TMPRSS6 antibodies. [Table 12]

[0196] As shown in Table 12, all of the selected TMPRSS6 antibodies inhibited the protease-dependent release of HJV from the cell surface by more than 90% in human TMPRSS6-expressing cells, with IC values ​​ranging from about 70 to about 200 pM. 50Two antibodies, REGN7999 and REGN8023, exhibited inhibitory effects in mouse TMPRSS6-expressing cells, with approximately 98.5% and approximately 42% inhibition of mouse TMPRSS6 protease activity and IC values ​​of approximately 82.6 pM and approximately 274 pM, respectively. 50 The values ​​were shown.

[0197] Table 13 shows the inhibition of cell surface HJV expression by anti-TMPRSS6 antibodies. [Table 13]

[0198] As shown in Table 13, all of the selected TMPRSS6 antibodies prevented the cleavage of HJV from the surface of HEK293.hHJV / 3xMyc.hTMPRSS6 cells. REGN7999 and REGN8023 also prevented the cleavage of HJV from the surface of HEK293.hHJV / 3xMyc.mTMPRSS6 cells.

[0199] Example 7: Inhibition of TMPRSS6 enzyme activity Experimental procedure Two bioassays were established to evaluate the inhibition of TMPRSS6 enzyme activity by anti-TMPRSS6 antibodies. In both bioassays, anti-TMPRSS6 antibodies or IgG subclass controls were incubated in assay buffer (PBS + Ca). ++ and Mg ++ ) from 500 nM to 122.1 pM (plus sample containing assay buffer alone without the test molecule).

[0200] The first bioassay ("CM bioassay") was performed with the extracellular domain of TMPRSS6 produced as conditioned medium (CM). CM was generated by engineering CHO cells to produce and secrete the extracellular domain of human TMPRSS6 (amino acids 68-802 of NCBI reference sequence NM_001289001) or cynomolgus TMPRSS6 (amino acids 66-800, V673M of NCBI reference sequence XM_005567384), both fused to 8x histidines at the N-terminus. For the CM bioassay, antibodies were incubated with either 0.2% hTMPRSS6 CM or 0.75% mfTMPRSS6 CM for 30 minutes at 25°C, and all CM percentages are expressed as v / v%.

[0201] The second bioassay ("HEK293 bioassay") was performed using HEK293 / hTMPRSS6, HEK293 / mfTMPRSS6, and HEK293 / mTMPRSS6 cell lines, which stably express TMPRSS6 from human, cynomolgus monkey, and mouse, respectively. Details regarding the preparation of these cell lines can be found in Example 5. For the HEK293 bioassay, cells were seeded at 10,000 cells / well (30 min preincubation) and 20,000 cells / well (overnight preincubation) in 96-well plates in DMEM high glucose + 10% FBS + Pen / Strep / L-glutamine (complete DMEM) and incubated at 37°C with 5% CO2. For the HEK293 cell bioassay using the 30 min pre-incubation condition, the day after seeding the cells, media was removed from the cells, all cells were washed once in assay buffer, and antibodies were added to the cells and incubated for 30 min at 25° C. For the overnight pre-incubation condition, the day after seeding the cells, antibodies were added to the cells, without washing, while still in complete DMEM, and incubated overnight at 37° C. with 5% CO2. The following day, media (containing antibodies) was removed from the cells and all cells were washed once in assay buffer.

[0202] For all assays, at the end of antibody / TMPRSS6 incubation and plate washing, 150 uM fluorescent TMPRSS6 peptide substrate (Boc-Gln-Ala-Arg-AMC, Enzo BML-P237) dissolved in assay buffer was added to antibody-treated cells or CM / antibody mixtures and incubated for 1 hour at 37°C with 5% CO2. After 1 hour of incubation, fluorescence was measured using an excitation wavelength of 380 nm and an emission wavelength of 460 nm on an Envision plate reader (Perkin Elmer). Results were analyzed using nonlinear regression (4-parameter logistic) in Prism software (GraphPad) to determine IC 50 The value was obtained from the following equation:

number

[0203] In this equation, the RFU ベースライン ” is the fluorescence value from either a certain concentration of TMPRSS6 CM or a certain number of HEK293 / TMPRSS6 cells without antibody, and “RFU” is the fluorescence value from either a certain concentration of TMPRSS6 CM or a certain number of HEK293 / TMPRSS6 cells without antibody, 阻害 " is the fluorescence value for the highest concentration of a particular antibody using the aforementioned concentration of TMPRSS6 CM or a particular number of HEK293 / TMPRSS6 cells, and "RLU バックグラウンド " is the fluorescence value without TMPRSS6 CM or HEK293 / TMPRSS6 cells.

[0204] result Seven anti-TMPRSS6 antibodies were selected and tested for their ability to inhibit the enzyme activity of TMPRSS6 CM and HEK293 / TMPRSS6 cells. Tables 14 to 16 show the results of these experiments. [Table 14]

[0205] As shown in Table 14, all of the selected antibodies exhibited inhibition of about 0.2% hTMPRSS6 CM ranging from about 65% to about 98% with ICs ranging from about 710 pM to greater than about 10 nM. 50 Additionally, all of the antibodies disclosed herein inhibited about 0.75% mfTMPRSS6 CM in the range of about 66% to about 98% with IC values ​​ranging from about 740 pM to greater than about 50 nM. 50 The values ​​are shown. [Table 15]

[0206] As shown in Table 15, when the antibodies were pre-incubated with 10,000 HEK293 / TMPRSS6 cells for 30 minutes, all of the selected antibodies exhibited HEK293 / hTMPRSS6 cell inhibition ranging from about 86% to about 90%, with IC ranging from about 360 pM to about 5.8 nM. 50 All of the selected antibodies showed inhibition of HEK293 / mfTMPRSS6 cells ranging from about 81% to about 94%, with IC values ​​ranging from about 660 pM to about 30 nM. 50 Three of the selected antibodies (REGN7977, REGN7999, and REGN8023) showed inhibition of HEK293 / mTMPRSS6 cells ranging from about 58% to about 88%, and the IC values ​​of these inhibitory antibodies were 50 The values ​​ranged from about 1.5 nM to about 35 nM. [Table 16]

[0207] As shown in Table 16, all of the selected antibodies inhibited HEK293 / hTMPRSS6 cells by about 86% to about 90%, with IC values ​​ranging from about 1.1 nM to about 2.6 nM, when the antibodies were preincubated overnight with 20,000 HEK293 / TMPRSS6 cells. 50 Additionally, all of the selected antibodies exhibited inhibition of HEK293 / mfTMPRSS6 cells ranging from about 31% to about 91%, with IC values ​​ranging from about 2.8 nM to greater than about 10 nM. 50Three of the selected antibodies (REGN7977, REGN7999, and REGN8023) showed inhibition of HEK293 / mTMPRSS6 cells ranging from about 97% to about 103%, and the IC values ​​of these inhibitory antibodies were 50 Values ​​ranged from about 1.2 nM to about 14 nM. An IgG subclass control antibody showed no measurable TMPRSS6 inhibition in any of the formats tested.

[0208] Example 8: Epitope mapping for human TMPRSS6 by HDX-MS Experimental procedure Hydrogen-deuterium exchange mass spectrometry (HDX-MS) was performed to determine the amino acid residues of human TMPRSS6 that interact with the TMPRSS6 monoclonal antibody subsets of the present disclosure, REGN7999 and REGN8023. HDX-MS methods are outlined, for example, in Ehring, Analytical Biochemistry (1999) 267(2):252-259, and Engen and Smith, Anal. Chem. (2001) 73:256A-265A.

[0209] HDX-MS experiments were performed on a customized platform, consisting of a custom HDX automation system for deuterium labeling and quenching, a Waters Acquity Binary Solvent Manager for sample digestion and loading, another Waters Acquity Binary Solvent Manager for the analytical gradient, and a Thermo Q Exactive HF mass spectrometer for peptide identification and mass measurement.

[0210] DO labeling solution was prepared in pD7.0 DO as PBS buffer (10 mM phosphate buffer, 140 mM NaCl, and 3 mM KCl, equivalent to pH 7.4 at 25° C.). For deuterium labeling, 10 μL of recombinant human TMPRSS6 extracellular domain expressed with S762A mutation and a C-terminal myc-myc-hexahistidine tag (hTMPRSS6_S762A-MMH; designated REGN5330; SEQ ID NO:117), premixed with either REGN7999 or REGN8023, was incubated in duplicate with 90 μL of DO labeling solution at 20° C. for various time points (non-deuterated control at 0 min, deuterium labeling for 5 or 10 min). 90 μL of quench buffer (0.5 M TCEP-HCl, 4 M urea, and 0.5% formic acid) was added to each sample to quench the deuteration reaction and incubated at 20° C. for 90 s. The quenched samples were then injected into the LC system for online pepsin / protease XIII digestion. The digested peptides were captured on a C18 column (2.1 mm×5 mm, Waters) and separated on another C18 column (2.1 mm×50 mm, Waters) with a 20 min gradient from 0% to 90% B at −5° C. (Mobile phase A: 0.5% formic acid and 4.5% acetonitrile in water; Mobile phase B: 0.5% formic acid in acetonitrile). The eluted peptides were analyzed by Thermo Q Exactive HF mass spectrometry in LC-MS / MS or LC-MS mode.

[0211] LC-MS / MS data from the non-deuterated hTMPRSS6_S762A-MMH sample were searched against a database containing the amino acid sequences of human TMPRSS6 protein, pepsin, protease XIII, and their reverse sequences using the Byonic search engine (Protein Metrics). Search parameters were set as default using non-specific enzymatic digestion and human glycosylation as common variable modifications. The list of identified peptides was then imported into HDExaminer software 3.1 (Sierra Analytics) to analyze the deuterium incorporation (D 取り込み) and deuterium incorporation percentage (D%) were calculated for the fully deuterated samples using the following formula:

number

[0212] result A total of 294 peptides from human TMPRSS6 were identified from both the hTMPRSS6_S762A-MMH samples alone and in complex with REGN7999, accounting for approximately 79.2% sequence coverage of human TMPRSS6. Peptides that showed a 5% or greater decrease in the percentage of deuterium incorporation were defined as significantly protected (ΔD%<-5%).

[0213] Table 17 shows human TMPRSS6 peptides with significant protection upon formation of the hTMPRSS6_S762A-MMH-REGN7999 complex compared to hTMPRSS6_S762A-MMH alone. Peptides corresponding to amino acids 693-703 SHFFEPGLHCW (SEQ ID NO: 120) and 780-805 LVSWGLGCGRPNYFGVYTRITGVISW (SEQ ID NO: 121) on human TMPRSS6 were significantly protected by REGN7999. [Table 17]

[0214] A total of 302 peptides from human TMPRSS6 were identified from both the hTMPRSS6 S762A-MMH alone and in complex with REGN8023 samples, representing approximately 79.6% sequence coverage of human TMPRSS6. Peptides that showed a 5% or greater decrease in percentage of deuterium incorporation were defined as significantly protected (ΔD%<-5%).

[0215] Table 18 shows human TMPRSS6 peptides with significant protection upon formation of hTMPRSS6 S762A-MMH-REGN8023 complex compared to hTMPRSS6 S762A-MMH alone. Peptides corresponding to amino acids 125-133 LITSTRLGT (SEQ ID NO: 122), 586-594 GEWPWQASL (SEQ ID NO: 123), 626-650 STVLWTVFLGKVWQNSRWPGEVSFK (SEQ ID NO: 124), and 704-724 ITGWGALREGGPISNALQKVD (SEQ ID NO: 125) on human TMPRSS6 were significantly protected by REGN8023. [Table 18-1] [Table 18-2]

[0216] Example 9: Epitope mapping for human TMPRSS6 by CryoEM Experimental procedure To further characterize the epitopes associated with the selected TMPRSS6 antibodies, cryo-electron microscopy (CryoEM) was used.

[0217] For preparation of Fab fragments, Fabricator enzyme (Genovis) was used according to the manufacturer's standard operating procedure to synthesize REGN7999 and REGN8023 IgG into F(ab') 2 and Fc fragments. F(ab') was purified using 2-mercaptoethylamine (2-MEA, ThermoFisher). 2The IgG was reduced to Fab, followed by removal of the Fc fragment using CaptureSelect IgG-Fc(ms) affinity resin (ThermoFisher). The Fab fragment was further purified by injection into a size-exclusion chromatography (SEC) column (Superdex 200 Increase 15 / 300 GL, GE healthcare) connected to an AKTA Avant 25 chromatography system (GE healthcare). The running buffer contained 25 mM Tris-HCl (pH 7.5), 150 mM NaCl. Peak fractions were pooled and concentrated with a 30 kDa cutoff centrifugal filter (Millipore Sigma) for use in subsequent complex preparations.

[0218] The human TMPRSS6 protein used in this experiment encompasses the extracellular domain residues G77-T811 of human TMPRSS6 fused to a C-terminal Myc-Myc-hexahistidine tag and contains a mutation in the catalytic domain (S762A) that renders the protein catalytically inactive (referred to as human TMPRSS6_S762A-MMH). For complex formation, 200 μg of purified human TMPRSS6_S762A-MMH was mixed with 250 μg each of REGN7999 Fab and REGN8023 Fab, then incubated on ice for approximately 30 min. The mixture was injected onto a SEC column (Superdex 200 Increase 15 / 300 GL, GE) connected to an AKTA Avant 25 chromatography system (GE healthcare). The SEC running buffer contained 25 mM Tris-HCl (pH 7.5), 150 mM NaCl. Fractions from a single monodisperse peak corresponding to the human TMPRSS6 S762A-MMH--REGN7999 Fab-REGN8023 Fab complex were pooled and concentrated with a 30 kDa cutoff centrifugal filter (Millipore Sigma) to a concentration of 3.5 mg / mL, as measured using a Nanodrop device (ThermoFisher).

[0219] CryoEM sample preparation and data collection were performed as follows: Freshly purified human TMPRSS6_S762A-MMH-REGN7999 Fab-REGN8023 Fab complex was diluted 2-fold to 1.75 mg / mL in buffer containing 25 mM Tris-HCl (pH 7.5), 150 mM NaCl for cryoEM grid preparation. n-Dodecyl-bD-maltopyranoside (Anatrace) was added to a final concentration of approximately 60 μM before immediately pipetting 3.5 μL of the mixture onto an UltrAufoil R1.2 / 1.3, 300 mesh grid (Quantifoil). Excess liquid was blotted using filter paper, and grids were flash frozen in liquid ethane cooled with liquid nitrogen using a Vitrobot Mark IV (ThermoFisher) (operated at 4°C and 100% humidity). The grid was then inserted into a Titan Krios G3i microscope (ThermoFisher) equipped with a K3 camera (Gatan). 3,580 movies were collected in counting mode at a nominal magnification of 81,000x (0.85 Å pixel size). Each movie contained 46 doses for a 2 s exposure, measuring Å. 2 The total acquired dose per was about 40 electrons.

[0220] CryoEM data processing and map generation were performed as follows: CryoEM data were first processed using Cryosparc v2.14.2. Movie motion was corrected by Patch motion collection, and CTF parameters were estimated by Patch CTF estimation. Particles were first picked using Blob picker to generate 2D class averages for template picking. 927,835 particles were picked using Template picker. After 2D classification, initial reconstruction, and heterogenous refinement, 94,036 particles were identified that correspond to the REGN5330-REGN7999 Fab-REGN8023 Fab complex. These particles were reconstructed at 3.4 Å resolution using heterogenous refinement. The resulting maps were used as the initial reference model for 3D classification in Relion. Maps used for model building were calculated using the Relion version 3.1 single particle processing pipeline. Movies were motion-corrected using MotionCor2 and CTF parameters were estimated using gctf. 3,411 parallelized microscopy images were selected for further processing based on having an estimated CTF resolution better than 4 Å. Using automated picking based on 2D templates, 1,445,534 particles were picked from the microscopy images and extracted as triply binned particle images (2.55 Å per pixel). Three rounds of 2D classification were performed to remove false positive complexes and truncated or incomplete complexes, resulting in 1,034,494 particles. These particles were subjected to 3D classification requiring eight classes using an initial reference model calculated with CryoSparc.270,021 particles from the two best classes, corresponding to the human TMPRSS6 S762A-MMH-REGN7999 Fab-REGN8023 Fab complex, were re-extracted as unbinned particle images and subjected to 3D refinement, which produced a map at 3.4 Å resolution, with the Fab and catalytic domains well resolved, but the remaining N-terminal domain of the protein poorly resolved. To better resolve the N-terminal domain, a focused classification was performed without alignment and with the application of a soft mask that excluded the Fab and catalytic domains. A single class containing 146,035 particles was identified as having improved density of the N-terminal domain. Refinement of these particles yielded a map at 3.6 Å resolution. After two rounds of CtfRefine and Bayesian polishing, three further rounds of focused 3D classification were performed without alignment and a soft mask around the CUB and SEA domains was applied, resulting in a final set of 105,142 particles. These particles were then refined to a 3.3 Å resolution (FSC=0.143) map with improved features of the N-terminal domain. For model building, the map was filtered at its local resolution value calculated in Relion, −76 Å. 2 The sharpening was performed to a B factor of 0.01.

[0221] Model building and refinement were performed as follows: manual model building was performed using Coot version 0.8.9, and real-space refinement was performed with Phenix version 1.17. A homology model of the S762A mutant of the human TMPRSS6 catalytic domain was generated from the published crystal structure of the human matriptase-1 catalytic domain (PDB 4IS5) using the Sculptor program in Phenix. This model was docked into the cryoEM map of the human TMPRSS6 S762A-MMH--REGN7999 Fab-REGN8023 Fab complex using Phenix AutoDock and manually adjusted. The LDLRa domain was manually built into the density using the published crystal structure of the LDLR domain (PDB 1AJJ) as a guide. Manual building of CUB domains 1 and 2 was guided by the published crystal structure of cubilin (PDB 3KQ4). Correct sequence register was demonstrated by the density of bulky residue side chains and N-linked glycosylation, as well as the location of disulfide bridges. The N-terminal SEA domain was not constructed due to insufficient fragmented density. The current model encompasses human TMPRSS6 residues 212-811, but excludes residues 573-581 and 662-664, which are located within the disordered linker. Homology models of the Fab fragments of REGN7999 and REGN8023 were generated from the previously determined REGN Fab structure. Unambiguous docking of the Fab models into the respective densities was facilitated by the clearly interpretable side chain densities corresponding to their distinct CDR sequences. After docking, the model was manually adjusted, followed by real-space refinement of the entire human TMPRSS6 S762A-MMH-REGN7999 Fab-REGN8023 Fab complex.

[0222] result CryoEM structure of human TMPRSS6 S762A-MMH-REGN7999 Fab-REGN8023 Fab complex: To better understand how anti-TMPRSS6 antibodies bind and inhibit TMPRSS6, a protein complex consisting of catalytically inactive human TMPRSS6 ectodomain (human TMPRSS6 S762A-MMH) bound to the Fab fragments of the anti-TMPRSS6 antibodies REGN7999 and REGN8023 was isolated by SEC and its three-dimensional structure was determined by single-particle cryo-EM. At a global resolution of 3.3 Å, the cryo-EM reconstruction was sufficient to build an atomic model of approximately 80% of the human TMPRSS6 ectodomain, encompassing its two CUB domains, three LDLRa domains, and the catalytic serine protease domain. The reconstruction also allowed for unambiguous docking of the Fab fragments of REGN7999 and REGN8023 and manual refitting of their variable regions. The local resolution at the paratope-epitope interface of both Fabs ranged from 3.1 to 3.5 Å, allowing precise determination of the interactions at the residue level between the antibodies and TMPRSS6. Furthermore, the structure of the tertiary complex confirms that REGN7999 and REGN8023 bind at non-overlapping epitopes.

[0223] Epitope and structural inhibitory mechanism of REGN7999: Residues that contribute to the REGN7999 epitope on TMPRSS6 are summarized in Table 18. These residues are defined as TMPRSS6 residues with non-hydrogen atoms within 4 Å of a non-hydrogen atom of REGN7999, and may involve hydrogen bonds, charge-charge interactions, or hydrophobic / van der Waals interactions. The REGN7999 epitope can be divided into two patches on TMPRSS6. The first patch is present in the catalytic domain and consists of TMPRSS6 residues G699, H701, D724, Q726, L727, I728, P729, L732, E735, G749, Y750, R751, K752, and N791. Contacts in this patch are mediated by a single residue from CDR H3, L2, L3, and framework region (FR) L3. The second patch is located on LDLRa repeat domain 2 and consists of residues V490, S501, T502, C503, I504, S505, and K508. The paratope of the second patch is composed of a continuous region of light chain residues that spans parts of CDR-L2 and FR-L3. The CUB and SEA domains are largely removed from the REGN7999 binding interface and do not contribute to its epitope on TMPRSS6. Data from hydrogen deuterium exchange (HDX) mass spectrometry experiments on REGN7999 (Example 8) are generally consistent with the catalytic domain patch observed in the CryoEM structure, but do not highlight residues from LDLRa domain 2 as contributing to the epitope.

[0224] Although its epitope includes a patch on the catalytic domain, REGN7999 does not contact the catalytic triad (H617, D668, and S672) where cleavage occurs, nor is it oriented to sterically block substrate access to the active site. The location of substrate bound to TMPRSS6 has not been experimentally determined, but this location can be inferred from the homologous structure of the matriptase serine protease domain complexed with a peptide inhibitor, reported in Yuan et al, BMC Struct Biol. (2011) 11:30. These structural observations suggest that binding of soluble hemojuvelin to the TMPRSS6 ectodomain is not perturbed by the presence of REGN7999. Without being bound by any theory, it is possible that REGN7999 may inhibit TMPRSS6 by allosterically "trapping" TMPRSS6 in an inactive conformation that can bind but not cleave substrates.

[0225] Epitope and structural inhibitory mechanism of REGN8023: Residues that contribute to the REGN8023 epitope on TMPRSS6 are summarized in Table 19. These residues are defined as TMPRSS6 residues with non-hydrogen atoms within 4 Å of a non-hydrogen atom of REGN8023, and may involve hydrogen bonds, charge-charge interactions, or hydrophobic / van der Waals interactions. The REGN8023 epitope is present exclusively in the catalytic domain and consists of TMPRSS6 residues R597, R599, I601, D622, L710, R711, E712, G713, G714, P715, and I716. The epitope observed in this structure is generally consistent with data from HDX mass spectrometry experiments. The REGN8023 paratope is contained primarily within the heavy chain and includes CDRs H1, H2, H3, L1, L3, and FR-H3.

[0226] Based on the above model, REGN8023 binds to the catalytic domain of TMPRSS6 such that the heavy chain does not directly contact the catalytic triad but is in a position to block substrate access to the active site. Without being bound by any theory, REGN8023 may inhibit TMPRSS6 through steric hindrance of substrate binding to the active site. This is also suggested by the binding data showing that REGN8023 competes with soluble hemojuvelin for binding to TMPRSS6 ectodomain protein. [Table 19]

[0227] Example 10: Comparison of REGN7999 and REGN8023 in Female WT Mice Experimental procedure Two in vivo experiments were performed to analyze the effects of selected TMPRSS6 antibodies on serum hepcidin and serum iron. Female wild-type mice with humanized HJV were used in both studies (Study 1: 11-13 weeks old, and Study 2: 6-9 weeks old). In both studies, mice were pre-bled on day 7. In the first study (Study 1), the two selected TMPRSS6 antibodies, REGN7999 and REGN8023, as well as an isotype control antibody, were administered subcutaneously to mice at 10 mg / kg on days 0, 2, and 7 of the study. Mice were then euthanized on day 9 and terminal blood was collected for analysis. In the second study (Study 2), the same two TMPRSS6 antibodies, REGN7999 and REGN8023, as well as an isotype control antibody, were administered subcutaneously to mice at 25 mg / kg on days 0, 2, and 7 of the study. Mice were then euthanized on day 14 and terminal blood was collected for analysis.

[0228] From both studies, serum iron was measured from terminal blood using a Siemens ADVIA Chemistry XPT instrument. Serum hepcidin was measured from terminal blood using a Hepcidin-murine-compete ELISA kit (Intrinsic Lifesciences, Cat: HMC-001, Lot: 5142019) according to the manufacturer's instructions.

[0229] result Measurements of serum iron (expressed in ng / mL) and serum hepcidin (expressed in μg / dL) from Study 1 are shown in Table 20 and from Study 2 in Table 21. [Table 20]

[0230] As shown in Table 20, in Study 1, REGN7999 treatment significantly increased serum hepcidin and significantly decreased serum iron in healthy REGN7999-treated mice compared to isotype control-treated mice. REGN8023 treatment also increased serum hepcidin and decreased serum iron in healthy REGN8023-treated mice, but this was not statistically significant compared to isotype control-treated mice. In Table 20, all values ​​are mean ± SD, n=6-8 per group. One-way ANOVA; * p<0.05 vs. isotype control Ab. [Table 21]

[0231] As shown in Table 21, in Study 2, REGN7999 treatment significantly increased serum hepcidin and significantly decreased serum iron in healthy REGN7999-treated mice compared to isotype control-treated mice. REGN8023 treatment also increased serum hepcidin and decreased serum iron in healthy REGN8023-treated mice, but only serum iron concentrations reached statistical significance compared to isotype control-treated mice. In Table 21, all values ​​are mean ± SD, n=4-5 per group. One-way ANOVA; * p<0.05 vs. isotype control Ab.

[0232] Data from both studies suggest that selected anti-TMPRSS6 antibodies may block TMPRSS6 function, resulting in the observed effects on hepcidin and serum iron concentrations.

[0233] Example 11: Effect of anti-TMPRSS6 on liver iron in a mouse model of hemochromatosis To further explore the efficacy of the anti-TMPRSS6 antibody REGN7999 in a relevant disease model, a hereditary hemochromatosis type 1 study was conducted. Hereditary hemochromatosis is an iron overload disease caused by mutations in the HFE protein that result in inappropriately low hepcidin concentrations (Finberg et al. Blood (2011) 117(17):4590-4599).

[0234] Experimental procedure Male mice homozygous for the deletion of the mouse HFE gene (HFE - / - We used the mouse model of hereditary hemochromatosis type 1 (HFE). - / -Mice (n=5-7) were injected subcutaneously with 10 mg / kg isotype control or REGN7999 every week, except for the first week, when two injections were administered. A wild-type mouse control group was included and administered the isotype control. After 8 weeks, mice were euthanized using CO2 asphyxiation. Terminal blood was collected and serum hepcidin was measured using a Hepcidin Murine-Compete ELISA kit (Intrinsic Lifesciences, Cat: HMC-001, Lot: 02032020) according to the manufacturer's operating instructions. After the mice were sacrificed, their livers were flash frozen. These livers were later processed to measure liver iron content based on the method described by Torrance and Bothwell, S. Afr. J. Med. Sci. (1968) 33(1):9-11.

[0235] result Values ​​for both serum hepcidin (reported in ng / mL) and liver iron content (reported in μg / g dry weight) are shown in Table 22. [Table 22]

[0236] As shown in Table 22, after 8 weeks of treatment with REGN7999, HFE - / - Mice had significantly increased serum hepcidin (about 1631 ng / mL) compared to mice treated with isotype control (about 339.3 ng / mL). Also, as shown in Table 22, after 8 weeks of treatment with REGN7999, HFE - / - Mice had significantly lower liver iron content (approximately 618.7 μg / g dry weight) compared to mice treated with isotype control (approximately 771.1 μg / g dry weight). In Table 22, all values ​​are mean ± SD, n = 5-7 per group. One-way ANOVA; * p<0.05 vs. HFE+ / +isotype control; ** p<0.05 vs. HFE- / - isotype control.

[0237] Example 12: Effect of anti-TMPRSS6 in a mouse model of beta thalassemia To evaluate the efficacy of the two anti-TMPRSS6 antibodies disclosed herein, a study was performed in a mouse model of beta-thalassemia intermedia, an iron overload disorder associated with ineffective hematopoiesis and iron dysregulation.

[0238] It recapitulates beta-thalassemia intermediate in humans (Ginzburg, Blood (2011) 118(16):4321-4330), has a heterozygous deletion of the β1 and β2 genes, and expresses homozygous expression of human hemojuvelin (HJV) instead of mouse HJV in a C57BL / 6 / 129S6 background. th3 / + × HJV HumIn We have generated mice carrying Hbb th3 / + × HJV HumIn Mice (n=10-19) were injected subcutaneously every week with either REGN7999, REGN8023, or isotype control at 10 mg / kg, except in week 1, when two injections were administered. A wild-type (WT) mouse control group was included and administered the isotype control in a similar manner. After 8 weeks, mice were euthanized using CO2 asphyxiation and whole blood was collected by cardiac puncture into serology tubes with or without EDTA. Serum hepcidin was measured using a commercially available Hepcidin Murine-Compete ELISA kit (Intrinsic Lifesciences, Cat: HMC-001, Lot: 08232019) according to the manufacturer's instructions. Serum iron and a complete hematology panel including reticulocytes and mature red blood cells (RBCs) were measured using a Siemens ADVIA Chemistry XPT instrument and an Oxford Science GENESIS Hematology System.

[0239] Furthermore, after the mice were sacrificed, their livers and spleens were flash frozen until further processing. Liver samples were later processed to measure liver iron content based on the method described by Torrance and Bothwell, S. Afr. J. Med. Sci. (1968) 33(1):9-11. Spleens were minced and filtered through a 70 mm strainer into a tube containing 10 mL autoMACS® buffer (Miltenyi Biotec, Catalog: 130-091-221). To harvest bone marrow from femurs, the hip joint was excised from mice and placed into a tube containing 100 mL autoMACS® buffer (Miltenyi Biotec, Catalog: 130-091-376) supplemented with bovine serum albumin (BSA). The tube was quickly centrifuged (10 s, 400×g) to release the bone marrow into solution.

[0240] Bone marrow and spleen cells were centrifuged at 400×g for 10 min at 4° C., followed by incubation with anti-mouse CD45 magnetic beads (Miltenyi Biotec, Cat: 130-052-301) for 20 min on ice. This was followed by magnetic separation on a multi-24 column (Miltenyi Biotec, Cat: 130-095-691). The flow-through, enriched for both mature and precursor RBCs, was collected and incubated with anti-mouse CD16 / CD32 Fc block (BD Biosciences, clone 2.4G2, Cat: 553142) for 10 min. This was followed by a 30 minute incubation with the following commercially available antibodies (all rat anti-mouse): FITC anti-Ter119, clone TER-119, catalog: 557915; APC anti-CD44, clone IM7, catalog: 561862; APC-Cy7 anti-CD45, clone 30-F11, catalog: 557659; APC-Cy7 anti-CD11b, clone M1 / 70, catalog: 557657; and APC-Cy7 anti-Ly6G, clone 1A8, catalog: 560600, all from BD Biosciences.

[0241] AADvanced (Thermofisher, S10349) was added for 30 min to exclude dead cells from the analysis. All staining procedures were performed on ice and compensation controls were included using OneComp eBeads™ (Invitrogen, Cat: 01-1111-42). A panel of minus-One controls was created to correct gating of target populations. Samples were run on a CytoFLEX LX Flow Cytometer (Beckman-Coulter) using fluorescence minus-one-control to gate target populations.

[0242] As shown in Table 23, after 8 weeks of treatment with REGN7999 or REGN8023, Hbb th3 / + × HJV HumIn Mice were treated with isotype control and WT mice for serum hepcidin levels. th3 / + × HJV HumIn These were significantly increased in both the human and mouse models (approximately 1510 ng / mL and approximately 1454 ng / mL, respectively) compared with the human models (approximately 225 ng / mL and approximately 511 ng / mL, respectively).

[0243] Furthermore, after 8 weeks of treatment with REGN7999 or REGN8023, Hbb th3 / + × HJV HumIn In the isotype control treated group, there was a significant reduction in serum iron (approximately 50 μg / dL) compared to the isotype control (approximately 128 μg / dL, Table 23). th3 / + × HJV HumIn Mice accumulated more liver iron than WT mice. This increase was more pronounced in female mice (-466 μg / g to -985 μg / g dry weight) compared to male mice (-215 μg / g to -364 μg / g dry weight) (Table 23). Reduction in liver iron content in response to REGN7999 and REGN8023 was also more effective in female mice after 8 weeks of treatment. In Table 23, all values ​​are mean ± SD, n = 10-19 per group. One-way ANOVA; *p < 0.01 vs. WT isotype control; ** p<0.05 vs. Hbb th3 / + Isotype control. [Table 23]

[0244] Hbb th3 / + × HJV HumIn Administration of REGN7999 or REGN8023 in mice resulted in an increase in red blood cells (about 10.51 M / μL and about 10.18 M / μL, respectively) compared to mice treated with an isotype control (about 8.72 M / μL, Table 24). th3 / + × HJV HumIn Eight weeks of treatment with REGN7999 or REGN8023 in mice resulted in a reduction in splenic reticulocytes (from approximately 38% in isotype control to approximately 18%; Ter-119 + / CD44 high ) and elevated levels of mature RBCs in both the spleen and bone marrow (Ter-119 + / CD44 low ) indicating improved erythropoiesis and less need for secondary erythropoiesis in the spleen (Table 25). Spleen weight was also significantly reduced in Hbb treated with anti-TMPRSS6 REGN7999 (~0.142 g) or REGN8023 (~0.130 g) compared to the isotype control (~0.301 g). th3 / + × HJV HumIn A significant decrease in mice was also observed (Table 24). In Table 24, all values ​​are mean ± SD, n = 10-19 per group. One-way ANOVA; * p < 0.05 vs. WT isotype control; ** p<0.05 vs. Hbb th3 / + Isotype control. In Table 25, all values ​​are mean ± SD, n = 10-19 per group. One-way ANOVA; * p<0.001 vs. WT isotype control; ** p<0.0001 vs. Hbb th3 / + Isotype control. [Table 24] [Table 25]

[0245] Example 13: Effect of REGN7999 in a mouse model of beta thalassemia To further evaluate the efficacy of REGN7999 disclosed herein, a study was performed in a mouse model of beta-thalassemia intermedia, as described in Example 12.

[0246] It recapitulates beta-thalassemia intermediate in humans (Ginzburg, Blood (2011) 118(16):4321-4330), has a heterozygous deletion of the β1 and β2 genes, and expresses homozygous expression of human hemojuvelin (HJV) instead of mouse HJV in a C57BL / 6 / 129S6 background. th3 / + × HJV HumIn We have generated mice carrying Hbb th3 / + × HJV HumIn Mice (n=7-9; see Example 12 for details regarding the generation of these mice) were injected subcutaneously with 5 mg / kg REGN7999 or isotype control every week (except that two injections were administered in week 1). A wild-type (WT) mouse control group was included and administered the isotype control in a similar manner. After 8 weeks, mice were treated and samples were collected as described in Example 12. Serum hepcidin, serum iron, and a complete hematology panel including reticulocytes and mature red blood cells were measured as described in Example 12.

[0247] Additionally, after the mice were sacrificed, their livers were flash frozen and later processed as described in Example 12. Values ​​for both serum hepcidin (reported in ng / mL) and liver iron content (reported in μg / g dry weight) are shown in Table 26. [Table 26]

[0248] As illustrated in Table 26, after 8 weeks of treatment with 5 mg / kg REGN7999, Hbb th3 / + × HJV HumIn Mice were treated with isotype control and WT mice for serum hepcidin levels. th3 / + × HJV HumIn In both mice (about 376.8 ng / mL and about 713.1 ng / mL, respectively), serum iron was significantly increased (about 1781 ng / mL) compared to Hbb123 mice (about 376.8 ng / mL and about 713.1 ng / mL, respectively). Also, as shown in Table 26, treatment with REGN7999 significantly increased serum iron levels in Hbb123 mice treated with the isotype control. th3 / + × HJV HumIn In the isotype-treated control study, Hbb decreased from approximately 372 μg / g dry weight in wild-type mice to approximately 138 μg / dL in wild-type mice. th3 / + × HJV HumIn An increase in liver iron content was observed in mice to approximately 547 μg / g dry weight. th3 / + × HJV HumIn In mice, treatment with REGN7999 did not significantly reduce liver iron content compared to isotype control (-513.9 and -547.2 μg / g dry weight, respectively), but a significant reduction was observed compared to mice treated with the wild-type isotype control (Table 26). In Table 26, all values ​​are mean ± SD, n=7-9 per group; one-way ANOVA * p < 0.05 vs. WT isotype control; ** p<0.05 vs. Hbb th3 / + × HJV HumIn Isotype control. [Table 27]

[0249] As shown in Table 27, hematocrit, red blood cell count, and hemoglobin concentration were, as expected, significantly higher in Hbb mice treated with isotype control compared to wild type mice. th3 / + × HJVHumIn Hematocrit was lower in the wild-type mice than in the control mice treated with 5 mg / kg Hbb th3 / + × HJV HumIn Treatment with REGN7999 reduced Hbb expression by approximately 28% in mice. th3 / + × HJV HumIn No effect on hematocrit was observed in mice treated with isotype control compared to the control (Table 27). Red blood cells (reported in M / μL) decreased from approximately 9.52 in wild type mice to 1.01 in Hbb3+ treated with isotype control. th3 / + × HJV HumIn After 8 weeks of treatment with REGN7999, Hbb th3 / + × HJV HumIn In mice treated with the wild-type isotype control, the hemoglobin concentration was approximately 13 g / dL, whereas in mice treated with the wild-type isotype control, the hemoglobin concentration was approximately 13 g / dL, whereas in mice treated with the wild-type isotype control, the hemoglobin concentration was approximately 13 g / dL. th3 / + × HJV HumIn Treatment with isotype control in mice significantly reduced hemoglobin to approximately 9.22 g / dL. Treatment with REGN7999 reduced Hbb th3 / + × HJV HumIn Increased hemoglobin concentrations to approximately 10.1 g / dL after 8 weeks of treatment in mice. Without being bound by theory, these data support the idea of ​​restricting iron by modulating the hepcidin pathway having beneficial effects on red blood cells. In Table 27, all values ​​are mean ± SD, n = 6-9 per group; one-way ANOVA * p < 0.05 vs. WT isotype control; ** p<0.05 vs. Hbb th3 / + × HJV HumIn Isotype control.

[0250] Example 14: Pharmacokinetics (PK) and Pharmacodynamics (PD) in Cynomolgus Monkeys The objective of this study was to determine the pharmacokinetic (PK) properties and pharmacokinetic / pharmacodynamic (PK / PD) relationships of two anti-TMPRSS6 human monoclonal antibodies (mAbs), REGN7999 and REGN8023, administered as a single intravenous (IV) infusion to female cynomolgus monkeys.

[0251] Twenty-one female cynomolgus monkeys were assigned to one of six groups (n=3 or 4 animals per group; 5 mg / kg or 15 mg / kg dose groups), as summarized in Table 28. Animals received a single IV infusion of 5 mg / kg REGN7999, 5 mg / kg REGN8023, 15 mg / kg REGN7999, 15 mg / kg REGN8023, or 15 mg / kg isotype control antibody (Table 28). Blood samples for determination of total drug concentrations were collected from all animals pre-dose and at various times throughout the 56-day survival period. Serum total human mAb concentrations were determined using a qualified anti-human Fc enzyme-linked immunosorbent assay (ELISA). PK parameters were estimated using noncompartmental analysis (NCA). Blood samples for pharmacodynamic testing were collected at various time points throughout the study and analyzed using standard clinical chemistry (Beckman Coulter AU680 analyzer) and hematology (Avida 120 analyzer) analyses. Serum hepcidin was measured using a commercially available Hepcidin Murine-Compete ELISA kit (Intrinsic Lifesciences, Cat: HMC-001, Lot: 08232019) according to the manufacturer's instructions. [Table 28]

[0252] The PK profiles of the anti-TMPRSS6 monoclonal antibodies (mAbs) REGN7999 or REGN8023 are characterized by nonlinear kinetics with target-mediated clearance (TMC) at low concentrations, and linear dose-proportional kinetics consistent with saturation of the target-mediated elimination pathway at high mAb concentrations. Following a single IV dose, the concentration-time profiles of total REGN7999 and total REGN8023 were characterized by an initial short-term distribution phase, followed by a short-term linear beta elimination phase, and at low concentrations by a terminal nonlinear target-mediated elimination phase and a post-target-mediated elimination phase. No conclusive evidence of anti-drug antibody (ADA)-like effects on the profiles and no apparent effects on exposure were observed, and therefore no concentration values ​​were excluded from the analysis.

[0253] Pharmacokinetic parameters are listed in Table 29. Following single IV doses of 5 mg / kg and 15 mg / kg of antibody, REGN7999 achieved maximum serum concentrations (C) of approximately 120 μg / mL and approximately 410 μg / mL, respectively. max ) values ​​were observed for REGN8023, with C values ​​of approximately 162 and approximately 508, respectively. max The corresponding dose-normalized C values ​​were observed. max (C max The C values ​​for the anti-TMPRSS6 mAbs REGN7999 and REGN8023 with increasing IV dose were within approximately 1.4-fold. max A dose-proportional increase in mean t max (C max The time taken to reach the target concentration (Tc) was observed for all dose groups at the first measurement time point of approximately 0.5 hours.

[0254] Exposure to REGN7999 (AUC inf ) increased more than dose-proportionally, with the AUC inf / dose was approximately 1.6-fold greater after administration of the 5 mg / kg dose. Consistent with this, a greater systemic clearance (CL) was observed at the 5 mg / kg dose, due to a greater effect of TMC at lower concentrations. Exposure to REGN8023 increased proportionally with increasing dose, suggesting a more subtle effect of TMC at the doses studied. The terminal half-life (t 1 / 2 ) ranged from approximately 7 to 8 days across all dose groups. [Table 29]

[0255] The pharmacodynamic effects of REGN7999 and REGN8023 on serum hepcidin, serum iron, and transferrin saturation were followed throughout the 57-day study period, as shown in Table 30. In groups treated with REGN7999 or REGN8023 at 5 mg / kg or 15 mg / kg, mean serum iron decreased by approximately 60% from baseline serum iron concentrations by day 1 post-dose.

[0256] As shown in Table 31, transferrin saturation decreased by about 12% on day 1 from about 35% at baseline, representing an improvement in serum iron transport capacity. The PD effect on day 1 was independent of dose, suggesting saturation of TMPRSS6 binding at concentrations achieved on day 1 for both low and high doses of REGN7999 and REGN8023. The reduction in serum iron and transferrin saturation was maintained through at least 6 weeks after REGN7999 administration in both the 5 mg / kg and 15 mg / kg dose groups. In the 5 mg / kg REGN8023 dose group, the pharmacodynamic effect on serum iron began to return toward baseline approximately 1 week after IV administration and was no longer statistically different from the isotype control treatment by week 4. The 15 mg / kg REGN8023 dose group had a longer duration of pharmacodynamic effect than the 5 mg / kg REGN8023 group due to sustained high drug concentrations. Both serum iron and transferrin saturation returned to near baseline values ​​by day 57 in most animals.

[0257] As shown in Table 32, serum hepcidin concentrations were slightly increased following administration of REGN7999 or REGN8023, although large inter-animal variability was observed. [Table 30] [Table 31] [Table 32]

[0258] Example 15: Effect of REGN7999 on anaerobic capacity in a mouse model of beta thalassemia The effect of REGN7999 treatment on anaerobic capacity in a mouse model of beta-thalassemia was evaluated.

[0259] It recapitulates beta-thalassemia intermediate in humans (Ginzburg, Blood (2011) 118(16):4321-4330), has a heterozygous deletion of the β1 and β2 genes, and expresses homozygous expression of human hemojuvelin (HJV) instead of mouse HJV in a C57BL / 6 / 129S6 background. th3 / + × HJV HumIn We have generated mice carrying Hbb th3 / + × HJV HumIn Mice (Example 12 for further details regarding the generation of these mice).

[0260] Hbb th3 / + REGN7999 treatment in mice appears to result in an increase in smaller, healthier-looking red blood cells that turn over more slowly (see, for example, Example 13).However, it was unclear whether this would lead to functional improvement of red blood cells after REGN7999 treatment.For example, it was unclear whether these smaller, healthier red blood cells could deliver sufficient oxygen under anaerobic conditions, such as during exhaustive running.

[0261] To test this, Hbb th3 / + Mice were subjected to forced running studies (see study design shown in Figure 1). Prior to all experiments, mice were trained for 2 weeks on the belt of a 6-lane motorized treadmill (Series 8 Treadmill, IITC Life Science) supplied with a shocker plate. For exhaustion runs, the base speed (10 m / min) was increased by 5 m / min every 5 min. The maximum amount of distance traveled was determined at 60 min. Mice that remained inactive against the shocker unit or received 500 shocks were removed from the treadmill.

[0262] Hbb th3 / + Mice were observed to run significantly less distance than wild-type C57BL / 6 mice treated with an isotype control antibody (REGN1945), suggesting that these mice became fatigued faster (Figure 2A). th3 / + Mice were able to run similar distances to wild-type animals (Figure 2A), indicating that the improvement in red blood cell health observed with REGN7999 treatment is reflected in improved anaerobic capacity. Wild-type C57BL / 6 mice injected with REGN7999 showed no difference in running distance compared to isotype-treated mice, suggesting that REGN7999 improved the performance of non-Hbb th3 / + These results show that it does not improve the running ability of mice (Figure 3).

[0263] Lactate measurements were also collected. To determine lactate production, 15 mL of blood was collected from the tail vein after the exhaustive run. Lactate concentrations were determined using a lactate meter (Nova Biomedical) as described in Summermatter et al. Proc Natl Acad Sci USA. 2013;110(21):8738-43. Control Hbb th3 / + In mice, Hbb treated with REGN7999 th3 / +Despite running less distance than mice, they produced more lactate in their blood, further suggesting improved red blood cell health (Figure 2B).

[0264] Example 16: Effects of REGN7999 and Acvr2b(L79D)-Fc on hepatic iron and erythropoiesis efficacy Acvr2b(L79D)-Fc has been shown to improve ineffective hematopoiesis and increase hemoglobin in mice and humans (see, e.g., Cappellini et al. N Engl J Med. 2020; 382(13): 1219-31, Suragani et al. Blood. 2014; 123(25): 3864-72). As shown herein, blocking TMPRSS6 with REGN7999 appears to improve ineffective hematopoiesis and result in more and healthier red blood cells. The effect of REGN7999 treatment on hepatic iron and erythropoiesis efficacy was evaluated and compared to treatment with Acvr2b(L79D)-Fc in a mouse model of beta thalassemia.

[0265] To compare the effects of REGN7999 treatment (10 mg / kg) and Acvr2b(L79D)-Fc treatment (10 mg / kg), Hbb th3 / + Mice were administered one of the two molecules for 8 weeks. Serum hepcidin, serum iron, and liver iron content were measured as described in Example 10. After 8 weeks of treatment with either molecule, it was observed that REGN7999 treatment increased serum hepcidin and reduced serum iron and liver iron content, whereas Acvr2b(L79D)-Fc treatment had no effect on these parameters (Figure 4A-C). Acvr2b(L79D)-Fc increased body weight but had no effect on liver weight (Figure 5). REGN7999 treatment was shown to reduce spleen weight, whereas Acvr2b(L79D)-Fc treatment did not (Figure 6). Hbb th3 / + Control mice had significant splenomegaly, which was partially reversed by treatment with REGN7999. Acvr2b(L79D)-Fc treatment reduced Hbb th3 / +It did not cause any change in splenomegaly in the mice.

[0266] Blood count panels were also collected from treated animals. As seen in the previous examples, blood count panels demonstrated that REGN7999 treatment reduced Hbb th3 / + The results show that Acvr2b(L79D)-Fc treatment significantly increased the number of red blood cells in mice (Table 33). th3 / + REGN7999 treatment significantly increased hemoglobin but did not increase red blood cell counts compared to controls. However, REGN7999 treatment did show a significant increase in red blood cell counts (Table 33).

[0267] Flow cytometry was used to confirm this finding. Red blood cells isolated from spleen or bone marrow were incubated with mouse Fc blocker (anti-CD16 / 32, BD Biosciences) for 10 min, followed by incubation with fluorescein isothiocyanate (FITC) anti-Ter119, phycoerythrin (PE) anti-CD71, allophycocyanin (APC) anti-CD44 and APC-cyanine 7 (APC-Cy7) anti-CD45 / CD11b / Ly6G cocktail for 30 min (all antibodies from BD Biosciences). To exclude dead cells from the analysis, cells were stained with Sytox AADvanced (Thermofisher) for 30 min. All staining procedures were performed on ice. Compensation controls were performed using OneComp beads (Invitrogen) and prepared at the time of cell staining. A panel of minus-One controls was created to correct gating of target populations. All samples were measured on a CytoFLEX Flow Cytometer (Beckman-Coulter).

[0268] Flow cytometry data showed an increase in mature erythrocytosis in the spleen only in REGN7999-treated mice, but not in Acvr2b(L79D)-Fc-treated mice (Figure 7). REGN7999 treatment also showed a decrease in reticulocytes, which was not observed in Acvr2b(L79D)-Fc-treated mice (Figure 8). [Table 33]

[0269] Arrange

Table 34-1

Table 34-2

Table 34-3

Table 34-4

Table 34-5

Table 34-6

Table 34-7

Table 34-8

Table 34-9

Table 34-10

Table 34-11

Table 34-12

Table 34-13

Table 34-14

Table 34-15

Table 34-16

Table 34-17

Table 34-18

Claims

1. An isolated antibody or antigen-binding fragment thereof that specifically binds to transmembrane serine protease 6 (TMPRSS6) protein, wherein the antibody or antigen-binding fragment thereof binds to the catalytic domain of TMPRSS6 but does not bind to the catalytic triad of TMPRSS6.

2. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment maintains TMPRSS6 in an inactive conformation that can bind to a TMPRSS6 substrate but cannot cleave it.

3. The isolated antibody or antigen-binding fragment thereof of claim 2, wherein the TMPRSS6 substrate is hemojuvelin (HJV).

4. 4. The isolated antibody or antigen-binding fragment thereof of claim 3, wherein the antibody or antigen-binding fragment thereof competes with hemojuvelin (HJV) for binding to TMPRSS6.

5. 1. An isolated antibody or antigen-binding fragment thereof that specifically binds to a transmembrane serine protease 6 (TMPRSS6) protein, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR), wherein the HCDR3 has an amino acid sequence selected from the HCDR3 sequences set forth in Table 1.

6. (a) the HCDR1 domain has an amino acid sequence selected from SEQ ID NO: 4, 24, 34, 44, 64, 84, or 102; (b) the HCDR2 domain has an amino acid sequence selected from SEQ ID NO: 6, 26, 36, 46, 66, 86, or 104; (c) the HCDR3 domain has an amino acid sequence selected from SEQ ID NO: 8, 28, 38, 48, 68, 88, or 106; (d) the LCDR1 domain has an amino acid sequence selected from SEQ ID NO: 12, 52, 72, 92, or 110; (e) the LCDR2 domain has an amino acid sequence selected from SEQ ID NO: 14, 54, or 74; and (f) The antibody or antigen-binding fragment thereof of claim 5, wherein the LCDR3 domain has an amino acid sequence selected from SEQ ID NO: 16, 56, 76, 94, or 112.

7. The three heavy chain CDRs and the three light chain CDRs are (a) SEQ ID NO: 4 (HCDR1), SEQ ID NO: 6 (HCDR2), SEQ ID NO: 8 (HCDR3), SEQ ID NO: 12 (LCDR1), SEQ ID NO: 14 (LCDR2), and SEQ ID NO: 16 (LCDR3); (b) SEQ ID NO:24 (HCDR1), SEQ ID NO:26 (HCDR2), SEQ ID NO:28 (HCDR3), SEQ ID NO:12 (LCDR1), SEQ ID NO:14 (LCDR2), and SEQ ID NO:16 (LCDR3); (c) SEQ ID NO: 34 (HCDR1), SEQ ID NO: 36 (HCDR2), SEQ ID NO: 38 (HCDR3), SEQ ID NO: 12 (LCDR1), SEQ ID NO: 14 (LCDR2), and SEQ ID NO: 16 (LCDR3); (d) SEQ ID NO: 44 (HCDR1), SEQ ID NO: 46 (HCDR2), SEQ ID NO: 48 (HCDR3), SEQ ID NO: 52 (LCDR1), SEQ ID NO: 54 (LCDR2), and SEQ ID NO: 56 (LCDR3); (e) SEQ ID NO: 64 (HCDR1), SEQ ID NO: 66 (HCDR2), SEQ ID NO: 68 (HCDR3), SEQ ID NO: 72 (LCDR1), SEQ ID NO: 74 (LCDR2), and SEQ ID NO: 76 (LCDR3); (f) SEQ ID NO:84 (HCDR1), SEQ ID NO:86 (HCDR2), SEQ ID NO:88 (HCDR3), SEQ ID NO:92 (LCDR1), SEQ ID NO:14 (LCDR2), and SEQ ID NO:94 (LCDR3), or (g) The antibody or antigen-binding fragment thereof of claim 5, comprising one of SEQ ID NO: 102 (HCDR1), SEQ ID NO: 104 (HCDR2), SEQ ID NO: 106 (HCDR3), SEQ ID NO: 110 (LCDR1), SEQ ID NO: 14 (LCDR2), and SEQ ID NO: 112 (LCDR3).

8. The antibody or antigen-binding fragment thereof according to claim 5, comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 100 / 108.

9. The antibody of claim 5, comprising the heavy chain (HC) / light chain (LC) amino acid sequence pair of sequence numbers 114 / 116.

10. The antibody, (a) is a fully human monoclonal antibody; (b) Human TMPRSS6 has a dissociation constant (K) of less than about 21.2 nM at 25° C. and 37° C. as measured by surface plasmon resonance assay. Ｄ ) to join (c) monkey TMPRSS6 has a K of less than about 25.7 nM at 25° C. and 37° C. as measured by surface plasmon resonance assay. Ｄ Join with (d) Mouse TMPRSS6 has a K of less than about 703 nM at 25° C. and 37° C. as measured by surface plasmon resonance assay. Ｄ Join with (e) Binding to cells expressing human TMPRSS6 and EC ５０ is less than about 2.7 nM; (f) Binding to monkey TMPRSS6-expressing cells and EC ５０ is less than about 3.6 nM; (g) Binding to cells expressing mouse TMPRSS6 and EC ５０ is less than about 35 nM be, (h) inhibiting protease-dependent release of cell surface hemojuvelin in the presence of human TMPRSS6 with a percent inhibition of greater than about 90%, e.g., inhibiting human TMPRSS6; (i) inhibiting protease-dependent release of cell surface hemojuvelin in the presence of human TMPRSS6, e.g., inhibiting human TMPRSS6 and having an IC of less than about 200 pM ５０ Showing, (j) inhibiting protease-dependent release of cell surface hemojuvelin in the presence of mouse TMPRSS6 with a percent inhibition of greater than about 42%, e.g., inhibiting mouse TMPRSS6; (k) inhibiting protease-dependent release of cell surface hemojuvelin in the presence of mouse TMPRSS6, e.g., inhibiting mouse TMPRSS6 with an IC of less than about 274 pM ５０ Showing, (l) inhibiting human TMPRSS6 with a percent inhibition of greater than about 65% at 25°C and 37°C; (m) inhibits human TMPRSS6 at 25° C. and 37° C. with an IC of less than about 10 nM ５０ Showing, (n) inhibits monkey TMPRSS6 with a percent inhibition of greater than about 66% at 25° C. and 37° C.; (o) inhibited monkey TMPRSS6 at 25°C and 37°C with an IC of less than about 50 nM ５０ Showing, (p) inhibiting mouse TMPRSS6 with a percent inhibition of greater than about 58% at 25°C and 37°C; (q) inhibits mouse TMPRSS6 at 25° C. and 37° C. with an IC of less than about 35 nM ５０ Showing, (r) when administered to a subject in need thereof, reduces the serum iron concentration in said subject; (s) when administered to a subject in need thereof, increases serum hepcidin levels in said subject. (t) when administered to a subject in need thereof, increases the level of mature red blood cells in said subject; (u) when administered to a subject in need thereof, increases the level of mature red blood cells in the spleen and / or bone marrow of said subject; (v) when administered to a subject in need thereof, increases the hemoglobin concentration in said subject; or (w) when administered to a subject in need thereof, reduces transferrin saturation levels in the subject.

11. A pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof that binds to TMPRSS6 according to any one of claims 1 to 9, and a pharma- ceutically acceptable carrier or diluent.

12. An isolated polynucleotide molecule comprising a polynucleotide sequence encoding the HCVR or LCVR of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

13. A host cell comprising the polynucleotide of claim 12.

14. A method for producing an antibody or antigen-binding fragment thereof that specifically binds to TMPRSS6, comprising culturing a host cell described in claim 13 under conditions that allow production of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof so produced.

15. An antibody or antigen-binding fragment thereof that binds to TMPRSS6 according to any one of claims 1 to 9 for use in preventing or ameliorating at least one symptom or sign of a disease or disorder associated with iron overload in a subject in need thereof, In some cases, the disease or disorder is selected from the group consisting of congenital dyserythroplastic anemia, Diamond-Blackfan anemia, alpha thalassemia, beta thalassemia, beta thalassemia major, beta thalassemia intermediate, transfusion-dependent hemolytic anemia, transfusion-dependent hemolytic anemia due to pyruvate kinase deficiency, sideroblastic transfusion-dependent hemolytic anemia, myelodysplastic syndromes, myelodysplastic syndromes with ringed sideroblasts, sickle cell disease, polycythemia vera, hereditary hemochromatosis, chronic liver disease, alcohol-related chronic liver disease, hepatitis C, and autoimmune hepatitis.

Images