Patent Application
20250136718
The computer-readable Sequence Listing submitted on May 24, 2024 and identified as follows: 124,367 bytes ST.26 XML document file named “033399-8014 Sequence Listing.xml,” created May 24, 2024, is incorporated herein by reference in its entirety.
The present disclosure relates to antibodies and antigen-binding fragments that bind TMPRSS6, and treating disorders including disorders of iron metabolism and myeloproliferative neoplasms, using antibodies and antigen-binding fragments that bind TMPRSS6.
Type II transmembrane serine protease 6 (TMPRSS6) is encoded by the TMPRSS6 gene and primarily expressed in liver. The structure of TMPRSS6 includes a type II transmembrane domain, followed by a sea urchin sperm protein, enteropeptidase and agrin (SEA) domain, a stem region containing two complement factor C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein (CUB) domains and three low-density lipoprotein receptor (LDLR) class A repeats, and a C-terminal trypsin-like serine protease domain (Wang, C.-Y. et al., Front. Pharmacol. 2014. 5:114). Aliases for TMPRSS6 (EC 3.4.21) include: matriptase-2; transmembrane protease serine 6; membrane-bound mosaic serine proteinase matriptase-2; and MT2.
TMPRSS6 plays a significant role in iron homeostasis through the BMP-SMAD signaling pathway that regulates the expression of hepcidin, a hormone that controls iron absorption and mobilization from iron stores. Hepcidin (also known as: HAMP (hepcidin anti-microbial protein or peptide), encoded by HAMP in humans and non-human primates, and Hamp in mice and rats) regulates systemic iron homeostasis by controlling the functional activity of the sole iron efflux channel ferroportin. Hepcidin can lower plasma iron levels by binding to ferroportin and causing internalization and degradation of the complex, thereby preventing iron absorption at the small intestine and release of stored iron. Chronic elevation of hepcidin levels causes systemic iron deficiency, and hepcidin deficiency causes systemic iron overload.
TMPRSS6 negatively regulates the production of hepcidin through a transmembrane signaling pathway that is triggered by iron deficiency and suppresses HAMP activation (Du, X. et al., Science 2008. 320:1088-1092; Wang, C.-Y. et al., Front. Pharmacol. 2014. 5:114). Low blood iron levels trigger this pathway to reduce hepcidin production, which allows more iron from the diet to be absorbed through the intestines and transported out of storage sites into the bloodstream. In rats under acute iron deprivation, hepatic TMPRSS6 protein levels are upregulated, leading to suppressed hepcidin expression and production (Wang, C.-Y. et al., Front. Pharmacol. 2014. 5:114). Mutations throughout the TMPRSS6 molecule, and especially in the extracellular domain, have been identified in subjects with iron deficiency anemia, in particular iron-refractory iron deficiency anemia (IRIDA) that is unresponsive to oral iron treatment and only partially responsive to parenteral iron therapy (Wang, C.-Y. et al., Front. Pharmacol. 2014. 5:114). Loss-of-function mutations in TMPRSS6 in humans result in elevated levels of hepcidin and iron-deficiency anemia (Camaschella, C., N Engl Journal Med 2013. 168:24) as overproduction of hepcidin leads to defective iron absorption and utilization.
Iron overload disorders result when excess iron accumulates in tissues and organs to an extent that their normal functions are disrupted. Iron toxicity is a common complication of iron overload disorders, leading to high rates of mortality as a result of iron accumulation in major organs. β-thalassemia is an iron overload disorder that occurs when mutations in the HBB gene cause reduced or absent production of β-globin (beta globin) that lead to apoptosis of erythroblasts and a shortage of mature red blood cells, resulting in ineffective erythropoiesis that causes anemia and hyperabsorption of iron leading to iron toxicity. In patients with β-thalassemia, hepcidin is abnormally suppressed in relation to the patient's state of iron loading, creating a hepcidin deficiency that in turn allows excessive iron absorption and development of systemic iron overload. Ineffective erythropoiesis in other disorders such as MDS (myelodysplastic syndrome), dyserythropoietic anemia, sideroblastic anemia, is likewise characterized by low hepcidin leading to iron overload. Hemochromatosis, e.g., hemochromatosis type 1 or hereditary hemochromatosis is an iron overload disorder characterized by excess intestinal absorption of dietary iron and a pathological increase in total body iron stores. Current standards of care for treating iron overload disorders include blood transfusions for ineffective erythropoiesis that can further exacerbate iron overload, iron chelation with poor patient compliance, and phlebotomy or splenectomy to manage symptoms. Therapeutic approaches currently under development include gene therapy targeting the HBB gene, gene therapy and gene editing targeting other relevant genes, hepcidin mimetics, Fc-fusion proteins that target TGF superfamily ligands to inhibit SMAD signaling, antisense RNA drugs targeting TMPRSS6 (e.g., El-Beshlawy A., et al., Blood Cells, Molecules and Diseases 2019. 76:53-58), and iRNA drugs targeting TMPRSS6.
Polycythemia vera (PV) is a chronic myeloproliferative neoplasm with constitutively activated JAK2/STAT5 signaling pathway, resulting in increased red cell mass and erythroid hyperplasia. The primary cause of mortality is attributable to thrombotic complications owing to hyperviscosity of the blood. Potential downstream conditions when JAK2/STAT5 signaling is constitutively activated may include concurrent aberrant erythropoiesis, an inflammatory milieu, decreased systemic iron concentration, and potentially altered hypoxia responsiveness that may directly influence iron absorption in certain tissues, any or all of which may play a role in iron metabolism in PV. (Ginzburg, Y. Z. et al., Leukemia 2018. 32:2105-2116) Evidence suggests that systemic iron deficiency or erythroid-targeted iron restriction could be beneficial in reducing erythrocytosis and normalizing the hematocrit in PV.
The invention relates to novel antibodies and antigen-binding fragments thereof that bind TMPRSS6, and methods of making and using antibodies and antigen-binding fragments thereof that bind TMPRSS6.
The present disclosure provides anti-TMPRSS6 antibodies, nucleic acids encoding anti-TMPRSS6 antibodies, and methods of making and using anti-TMPRSS6 antibodies. Anti-TMPRSS6 antibodies as disclosed herein encompass anti-TMPRSS6 antibodies and fragments thereof that are capable of binding TMPRSS6. Anti-TMPRSS6 antibodies as disclosed herein are capable of binding to human TMPRSS6 on the surface of a cell expressing human TMPRSS6. The present disclosure provides anti-TMPRSS6 antibodies for therapeutic and diagnostic uses. Anti-TMPRSS6 antibodies as disclosed herein can be used to treat disorders of iron metabolism such as iron overload disorders, in particular β-thalassemias including but not limited to non-transfusion dependent thalassemia, and other disorders of ineffective erythropoiesis. Anti-TMPRSS6 antibodies as disclosed herein can be used to treat myeloproliferative disorders such as polycythemia vera (PV) characterized by erythrocytosis and erythroid hyperplasia.
In one aspect, anti-TMPRSS6 antibodies are provided that are capable of binding to TMPRSS6 on the surface of a cell expressing TMPRSS6 and modulating the activity of at least one component involved in iron metabolism, where a component may be a molecule or a biological process associated with the function of TMPRSS6. In certain embodiments, anti-TMPRSS6 antibodies disclosed herein are capable of modulating the activity of at least one component involved in regulating hepcidin expression. In certain embodiments, anti-TMPRSS6 antibodies disclosed herein are capable of substantially inhibiting TMPRSS6 suppression of hepcidin expression. In certain embodiments, anti-TMPRSS6 antibodies disclosed herein are capable of increasing hepcidin expression. In certain embodiments, anti-TMPRSS6 antibodies disclosed herein are capable of increasing the activity of the hepcidin promoter. In certain embodiments, anti-TMPRSS6 antibodies disclosed herein are capable of substantially inhibiting TMPRSS6 suppression of the BMP/SMAD pathway-induced expression of hepcidin. Anti-TMPRSS6 antibodies disclosed herein may modulate hepcidin expression, including but not limited to substantially inhibiting TMPRSS6 suppression of hepcidin expression, increasing hepcidin expression, increasing hepcidin promoter activity, or substantially inhibiting TMPRSS6 suppression of the BMP/SMAD pathway-induced expression of hepcidin, in a dose-dependent manner. In certain embodiments, anti-TMPRSS6 antibodies disclosed herein are capable of modulating hepcidin expression in a dose-dependent manner. In certain embodiments, anti-TMPRSS6 antibodies disclosed herein are capable of increasing serum hepcidin levels in a dose-dependent manner when administered to a subject. In certain embodiments, anti-TMPRSS6 antibodies disclosed herein are capable of reducing serum iron levels in a dose-dependent manner when administered to a subject. In certain embodiments, anti-TMPRSS6 antibodies disclosed herein are capable of increasing liver hepcidin RNA levels in a dose-dependent manner when administered to a subject. In certain embodiments, anti-TMPRSS6 antibodies disclosed herein are capable of reducing liver non-heme iron, increasing serum hepcidin, increasing liver hepcidin RNA, reducing splenomegaly, increasing red blood count (RBC), increasing hematocrit (HCT), reducing red cell distribution width (RDW), and increased production of mature red cells (increased erythropoiesis) when administered to a subject known or suspected to have an iron overload disorder, in particular a β-thalassemia. In certain embodiments, anti-TMPRSS6 antibodies disclosed herein are capable of reducing RBC, reducing HCT, reducing Hemoglobin (HGB), reducing mean corpuscular volume (MCV), and reducing RDW when administered to a subject known or suspected to have a myeloproliferative disorder, such as a myeloproliferative neoplasm, in particular polycythemia vera (PV).
In another aspect, anti-TMPRSS6 antibodies disclosed herein show cross-reactivity with at least one non-human TMPRSS6. In certain embodiments, anti-TMPRSS6 antibodies disclosed herein are capable of binding to at least one non-human TMPRSS6 on the surface of a cell expressing the at least one non-human TMPRSS6. Anti-TMPRSS6 antibodies disclosed herein may be capable of binding human TMPRSS6 and mouse TMPRSS6. Anti-TMPRSS6 antibodies disclosed herein may be capable of binding to human TMPRSS6 and cynomolgus monkey TMPRSS6. Anti-TMPRSS6 antibodies disclosed herein may be capable of binding to each of human TMPRSS6, mouse TMPRSS6, and cynomolgus monkey TMPRSS6.
In another aspect, anti-TMPRSS6 antibodies disclosed herein specifically bind to TMPRSS6 (matriptase-2). In certain embodiments, anti-TMPRSS6 antibodies disclosed herein bind to TMPRSS6 (matriptase-2) and do not show detectable binding to matriptase homologues. In certain embodiments, anti-TMPRSS6 antibodies disclosed herein bind to human TMPRSS6 (matriptase-2) and do not show detectable binding to human matriptase-1 (ST14). In certain embodiments, anti-TMPRSS6 antibodies disclosed herein bind to human TMPRSS6 (matriptase-2) and do not show detectable binding to human matriptase-3 (TMPRSS7). In certain embodiments, anti-TMPRSS6 antibodies disclosed herein bind to human TMPRSS6 (matriptase-2) and do not show detectable binding to either of human matriptase-1 (ST14) or human matriptase-3 (TMPRSS7).
An anti-TMPRSS6 antibody disclosed herein may be a monoclonal antibody, a humanized antibody, a chimeric antibody, a single chain antibody, a Fab fragment, a single-chain variable fragment (scFv), a recombinant antibody, a recombinant monoclonal antibody, an aptamer, a single-domain antibody (VHH, nanobody), or other TMPRSS6-binding fragment or variant. In certain embodiments, an anti-TMPRSS6 antibody disclosed herein may comprise a framework in which amino acids have been substituted into an existing antibody framework, in particular to influence properties such as antigen-binding ability. In certain embodiments, an anti-TMPRSS6 antibody disclosed herein may comprise complementarity determining regions (CDRs) from a source (parental) antibody that have been grafted (fused) into a framework from a different type (class) of antibody and/or from a different organism than the parental antibody, in particular an acceptor human framework. In certain embodiments, an anti-TMPRSS6 antibody disclosed herein may comprise a framework in which amino acids have been substituted, mutated, or replaced in regions outside of the CDRs to influence properties such as antigen-binding or antibody structure, e.g., in the variable region framework surrounding the CDRs and/or in a constant region, in particular the Fc region. In certain embodiments, one or more of the CDRs have been substituted, mutated, or replaced. In certain embodiments, an anti-TMPRSS6 antibody disclosed herein may be a humanized anti-TMPRSS6 antibody variant.
In certain embodiments, anti-TMPRSS6 antibodies disclosed herein comprise at least one polypeptide having an amino acid sequence as set forth in Table 1, Table 2, or Table 3, or a sequence substantially identical (e.g., at least 85%, 90%, 92%, 95%, 97%, or 98%, 99% identical) to an amino acid sequence as set forth in Table 1, Table 2, or Table 3. Anti-TMPRSS6 antibodies disclosed herein may comprise at least one polypeptide having an amino acid sequence selected from the following, or a sequence substantially identical (e.g., at least 85%, 90%, 92%, 95%, 97%, or 98%, 99% identical) to at least one polypeptide having an amino acid sequence selected from the following: SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8; SEQ ID NO: 9; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 17; SEQ ID NO: 18; SEQ ID NO: 19; SEQ ID NO: 21; SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID NO: 24; SEQ ID NO: 26; SEQ ID NO: 27; SEQ ID NO: 28; SEQ ID NO: 29; SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 33; SEQ ID NO: 34; SEQ ID NO: 36; SEQ ID NO: 37; SEQ ID NO: 38; SEQ ID NO: 39; SEQ ID NO: 41; SEQ ID NO: 42; SEQ ID NO: 43; SEQ ID NO: 44; SEQ ID NO: 46; SEQ ID NO: 47; SEQ ID NO: 48; SEQ ID NO: 49; SEQ ID NO: 51; SEQ ID NO: 52; SEQ ID NO: 53; SEQ ID NO: 54; SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 58; SEQ ID NO: 59; SEQ NO: 61; SEQ ID NO: 63; SEQ ID NO: 65; SEQ ID NO: 67; SEQ ID NO: 69; SEQ ID NO: 71; SEQ ID NO: 73; SEQ ID NO: 75; SEQ ID NO: 77; SEQ ID NO: 79; SEQ ID NO: 81; or SEQ ID NO: 83.
In one embodiment, an anti-TMPRSS6 antibody disclosed herein comprises a heavy chain (HC) variable region polypeptide of the amino acid sequence set forth in SEQ ID NO: 1 or a sequence substantially identical to SEQ ID NO: 1, and a light chain (LC) variable region polypeptide of the amino acid sequence set forth in SEQ ID NO: 6 or a sequence substantially identical to SEQ ID NO: 6. In one embodiment, an anti-TMPRSS6 antibody disclosed herein comprises a heavy chain complementarity determining region 1 (HC CDR1) having the amino acid sequence GYTFTSYW set forth in SEQ ID NO: 2, a heavy chain complementarity determining region 2 (HC CDR2) having the amino acid sequence IYPGSGST set forth in SEQ ID NO: 3, a heavy chain complementarity determining region 3 (HC CDR3) having the amino acid sequence APYDSDYAMDY set forth in SEQ ID NO: 4; a light chain complementarity determining region 1 (LC CDR1) having the amino acid sequence QDINNY set forth in SEQ ID NO: 7, a light chain complementarity determining region 2 (LC CDR2) having the amino acid sequence RAN set forth in SEQ ID NO: 8, and a light chain complementarity determining region 3 (LC CDR3) having the amino acid sequence LQYDEFPLT set forth in SEQ ID NO: 9; or a variant of said antibody comprising 1, 2, 3, 4, 5, or 6 amino acid substitutions in the CDR regions. In one non-limiting embodiment, an anti-TMPRSS6 antibody disclosed herein is the antibody identified herein as MWTx-001, comprising an HC polypeptide having the amino acid sequence set forth in SEQ ID NO: 61 and an LC polypeptide having the amino acid sequence set forth in SEQ ID NO: 63.
In one embodiment, an anti-TMPRSS6 antibody disclosed herein comprises an HC variable region polypeptide of the amino acid sequence set forth in SEQ ID NO: 11 or a sequence substantially identical to SEQ ID NO: 11, and an LC variable region polypeptide of the amino acid sequence set forth in SEQ ID NO: 16 or a sequence substantially identical to SEQ ID NO: 16. In one embodiment, an anti-TMPRSS6 antibody disclosed herein comprises an HC CDR1 having the amino acid sequence GFNIKDYY set forth in SEQ ID NO: 12, an HC CDR2 having the amino acid sequence IDPEDGES set forth in SEQ ID NO: 13, an HC CDR3 having the amino acid sequence TRGDSMMVTYFDY set forth in SEQ ID NO: 14; an LC CDR1 having the amino acid sequence QDVSTA set forth in SEQ ID NO: 17, an LC CDR2 having the amino acid sequence WAF set forth in SEQ ID NO: 18, and an LC CDR3 having the amino acid sequence QQHYRSPWT set forth in SEQ ID NO: 19, or a variant of said antibody comprising 1, 2, 3, 4, 5, or 6 amino acid substitutions in the CDR regions. In one non-limiting embodiment, an anti-TMPRSS6 antibody disclosed herein is of the antibody identified herein as MWTx-002, comprising an HC polypeptide having the amino acid sequence set forth in SEQ ID NO: 65 and an LC polypeptide having the amino acid sequence set forth in SEQ ID NO: 67.
In one embodiment, an anti-TMPRSS6 antibody disclosed herein comprises an HC variable region polypeptide of the amino acid sequence set forth in SEQ ID NO: 21 or a sequence substantially identical to SEQ ID NO: 21, and an LC variable region polypeptide of the amino acid sequence set forth in SEQ ID NO: 26 or a sequence substantially identical to SEQ ID NO: 26. In one embodiment, an anti-TMPRSS6 antibody disclosed herein comprises an HC CDR1 having the amino acid sequence GFNIEDYY set forth in SEQ ID NO: 22, an HC CDR2 having the amino acid sequence IDPEDGET set forth in SEQ ID NO: 23, an HC CDR3 having the amino acid sequence ARSIYLDPMDY set forth in SEQ ID NO: 24; an LC CDR1 having the amino acid sequence QDVTTA set forth in SEQ ID NO: 27, an LC CDR2 having the amino acid sequence WAT set forth in SEQ ID NO: 28, and an LC CDR3 having the amino acid sequence QQHYSTPYT set forth in SEQ ID NO: 29, or a variant of said antibody comprising 1, 2, 3, 4, 5, or 6 amino acid substitutions in the CDR regions. In one non-limiting embodiment, an anti-TMPRSS6 antibody disclosed herein is the antibody identified herein as MWTx-003, comprising an HC polypeptide having the amino acid sequence set forth in SEQ ID NO: 69 and an LC polypeptide having the amino acid sequence set forth in SEQ ID NO: 71.
In one embodiment, an anti-TMPRSS6 antibody disclosed herein comprises an HC variable region polypeptide of the amino acid sequence set forth in SEQ ID NO: 31 or a sequence substantially identical to SEQ ID NO: 31, and an LC variable region polypeptide of the amino acid sequence set forth in SEQ ID NO: 36 or a sequence substantially identical to SEQ ID NO: 36. In one embodiment, an anti-TMPRSS6 antibody disclosed herein comprises an HC CDR1 having the amino acid sequence GYTFTSYW set forth in SEQ ID NO: 32, an HC CDR2 having the amino acid sequence IYPGSGST set forth in SEQ ID NO: 33, an HC CDR3 having the amino acid sequence APYDADYAMDY set forth in SEQ ID NO: 34; an LC CDR1 having the amino acid sequence QDISNY set forth in SEQ ID NO: 37, an LC CDR2 having the amino acid sequence RAN set forth in SEQ ID NO: 38, and an LC CDR3 having the amino acid sequence LQYDEFPLT set forth in SEQ ID NO: 39, or a variant of said antibody comprising 1, 2, 3, 4, 5, or 6 amino acid substitutions in the CDR regions. In one non-limiting embodiment, an anti-TMPRSS6 antibody disclosed herein is the antibody identified herein as humanized anti-TMPRSS6 antibody variant hzMWTx-001 Var, comprising an HC polypeptide having the amino acid sequence set forth in SEQ ID NO: 73 and an LC polypeptide having the amino acid sequence set forth in SEQ ID NO: 75.
In one embodiment, an anti-TMPRSS6 antibody disclosed herein comprises an HC variable region polypeptide of the amino acid sequence set forth in SEQ ID NO: 41 or a sequence substantially identical to SEQ ID NO: 41, and an LC variable region polypeptide of the amino acid sequence set forth in SEQ ID NO: 46 or a sequence substantially identical to SEQ ID NO: 46. In one embodiment, an anti-TMPRSS6 antibody disclosed herein comprises an HC CDR1 having the amino acid sequence GFNIKDYY set forth in SEQ ID NO: 42, an HC CDR2 having the amino acid sequence IDPEDAES set forth in SEQ ID NO: 43, an HC CDR3 having the amino acid sequence TRGDSMMVTYFDY set forth in SEQ ID NO: 44; an LC CDR1 having the amino acid sequence QDVSTA set forth in SEQ ID NO: 47, an LC CDR2 having the amino acid sequence WAF set forth in SEQ ID NO: 48, and an LC CDR3 having the amino acid sequence QQHYRSPWT set forth in SEQ ID NO: 49, or a variant of said antibody comprising 1, 2, 3, 4, 5, or 6 amino acid substitutions in the CDR regions. In one non-limiting embodiment, an anti-TMPRSS6 antibody disclosed herein is the antibody identified herein as humanized anti-TMPRSS6 antibody variant hzMWTx-002 Var, comprising an HC polypeptide having the amino acid sequence set forth in SEQ ID NO: 77 and an LC polypeptide having the amino acid sequence set forth in SEQ ID NO: 79.
In one embodiment, an anti-TMPRSS6 antibody disclosed herein comprises an HC variable region polypeptide of the amino acid sequence set forth in SEQ ID NO: 51 or a sequence substantially identical to SEQ ID NO: 51, and an LC variable region polypeptide of the amino acid sequence set forth in SEQ ID NO: 56 or a sequence substantially identical to SEQ ID NO: 56. In one embodiment, an anti-TMPRSS6 antibody disclosed herein comprises an HC CDR1 having the amino acid sequence GFNIEDYY set forth in SEQ ID NO: 52, an HC CDR2 having the amino acid sequence IDPEDAET set forth in SEQ ID NO: 53, an HC CDR3 having the amino acid sequence ARSIYLDPMDY set forth in SEQ ID NO: 54; an LC CDR1 having the amino acid sequence QDVTTA set forth in SEQ ID NO: 57, an LC CDR2 having the amino acid sequence WAT set forth in SEQ ID NO: 58, and an LC CDR3 having the amino acid sequence QQHYSTPYT set forth in SEQ ID NO: 59, or a variant of said antibody comprising 1, 2, 3, 4, 5, or 6 amino acid substitutions in the CDR regions. In one non-limiting embodiment, an anti-TMPRSS6 antibody disclosed herein is the antibody identified herein as humanized anti-TMPRSS6 antibody variant hzMWTx-003 Var, comprising an HC polypeptide having the amino acid sequence set forth in SEQ ID NO: 81 and an LC polypeptide having the amino acid sequence set forth in SEQ ID NO: 83.
In another aspect, anti-TMPRSS6 antibodies (including variants and fragments as disclosed herein) are provided that can be used to treat disorders of iron metabolism such as iron overload disorders, in particular β-thalassemia and other disorders of ineffective erythropoiesis. Methods and compositions are provided for using anti-TMPRSS6 antibodies as disclosed herein for therapeutic uses including, but not limited to, treating disorders of iron metabolism such as iron overload disorders, in particular β-thalassemia and other disorders of ineffective erythropoiesis. In certain embodiments, pharmaceutical compositions comprising an anti-TMPRSS6 antibody disclosed herein and a suitable carrier and/or excipient are provided.
In another aspect, methods for treating a disorder of iron metabolism are provided, such methods comprising administering an effective amount of an anti-TMPRSS6 antibody disclosed herein to a subject in need thereof, wherein administration of the effective amount of anti-TMPRSS6 antibody modulates the activity of a component involved in iron metabolism. In certain embodiments, methods for treating an iron overload disorder comprise administering an effective amount of an anti-TMPRSS6 antibody disclosed herein, wherein administration of the effective amount of anti-TMPRSS6 antibody modulates the activity of a component involved in iron metabolism. In certain embodiments, methods for treating an iron overload disorder comprise administering an effective amount of an anti-TMPRSS6 antibody disclosed herein, wherein administration of the effective amount of anti-TMPRSS6 antibody modulates the activity of at least one component involved in regulating hepcidin expression. In certain embodiments, methods comprise administration of an effective amount of anti-TMPRSS6 antibody that inhibits TMPRSS6 suppression of hepcidin expression. In certain embodiments, administration of the effective amount of anti-TMPRSS6 antibody increases hepcidin expression. In certain embodiments, methods comprise administration of an effective amount of anti-TMPRSS6 antibody that increases the activity of the hepcidin promoter. In certain embodiments, methods comprise administration of an effective amount of anti-TMPRSS6 antibody that inhibits TMPRSS6 suppression of the BMP/SMAD pathway-induced expression of hepcidin. In certain embodiments, methods comprise administration of an effective amount of anti-TMPRSS6 antibody to a subject that results in one or more biological effects associated with an iron overload disorder including but not limited to reducing serum iron, reducing liver non-heme iron, increasing serum hepcidin, increasing liver hepcidin RNA, reducing splenomegaly, increasing red blood count (RBC), increasing hematocrit (HCT), reducing red cell distribution width (RDW), and/or increased production of mature red cells (increased erythropoiesis).
In another aspect, methods for treating a disease or disease state in which abnormal suppression of hepcidin expression is involved are provided, such methods comprising administering an effective amount of an anti-TMPRSS6 antibody disclosed herein to a subject in need thereof, wherein administration of the effective amount of anti-TMPRSS6 antibody modulates the activity of at least one component involved in abnormal suppression of hepcidin expression and reduces abnormal suppression of hepcidin expression. In particular embodiments, the method results in increased hepcidin expression.
In another aspect, methods for treating a disorder of iron metabolism associated with suppressed hepcidin levels are provided, such methods comprising administering an effective amount of an anti-TMPRSS6 antibody disclosed herein to a subject in need thereof, wherein administration of the effective amount of anti-TMPRSS6 antibody modulates the activity of at least one component involved in suppression of hepcidin levels. In certain embodiments, methods comprise administration of an effective amount of anti-TMPRSS6 antibody that increases serum hepcidin levels, increases liver hepcidin RNA, and lowers serum iron levels.
In another aspect, methods are provided for treating disorders of iron metabolism including disorders related to and/or characterized by ineffective erythropoiesis that may include but are not limited to β-thalassemia. In accordance with this aspect, such methods comprise administering an effective amount of an anti-TMPRSS6 antibody disclosed herein to a subject that is known or suspected of having a disorder of iron metabolism related to and/or characterized by ineffective erythropoiesis, wherein administration results in one or more changes related to iron metabolism and/or erythropoiesis in the subject. In certain embodiments, methods are provided wherein administration of the effective amount of anti-TMPRSS6 antibody treats or ameliorates at least one biological effect or symptom associated with the disorder. In particular embodiments, practicing the method results in one or more changes including but not limited to reducing liver non-heme iron, increasing serum hepcidin, increasing liver hepcidin RNA, reducing splenomegaly, increasing red blood count (RBC), increasing hematocrit (HCT), reducing red cell distribution width (RDW), and increased production of mature red cells (increased erythropoiesis).
In another aspect, methods are provided for treating a myeloproliferative disorder, including but not limited to myeloproliferative neoplasm, myeloproliferative neoplasm with constitutively activated JAK2/STAT5 signaling pathway, myeloproliferative disorders characterized by increased red cell mass and erythroid hyperplasia, polycythemia vera (PV), and/or disorders characterized by erythrocytosis and erythroid hyperplasia. In accordance with this aspect, such methods comprise administering an effective amount of an anti-TMPRSS6 antibody disclosed herein to a subject that is known or suspected of having a myeloproliferative disorder. In certain embodiments, methods are provided wherein administration of the effective amount of anti-TMPRSS6 antibody treats or ameliorates at least one biological effect or symptom associated with the disorder. In particular embodiments, practicing the method results in one or more changes including but not limited to reducing RBC, reducing HCT, reducing hemoglobin (HGB), reducing mean corpuscular volume (MCV), and reducing RDW when administered to a subject known or suspected to have a myeloproliferative disorder. In a particular embodiment, practicing the method results in one or more changes including but not limited to reducing RBC, reducing HCT, reducing hemoglobin (HGB), reducing mean corpuscular volume (MCV), and reducing RDW when administered to a subject known or suspected to have polycythemia vera (PV).
In another aspect, methods for diagnosing or screening for an iron overload disorder in a subject are provided. In certain embodiments, methods comprise administering anti-TMPRSS6 antibody to a subject known or suspected to have an iron overload disorder and measuring one or more biological effect or symptom associated with an iron overload disorder.
In another aspect, methods for diagnosing or screening for a myeloproliferative disorder in a subject are provided. In certain embodiments, methods comprise administering anti-TMPRSS6 antibody to a subject known or suspected to have a myeloproliferative disorders and measuring one or more biological effect or symptom associated with a myeloproliferative disorder.
In another aspect, one or more isolated nucleic acid molecules are provided that encode at least a portion of at least one of the anti-TMPRSS6 antibodies disclosed herein. In certain embodiments, isolated nucleic acid molecules that encode at least a portion of at least one of the anti-TMPRSS6 antibodies disclosed herein comprise a nucleotide sequence as set forth in Table 1, Table 2, or Table 3, or a sequence substantially identical (e.g., at least 85%, 90%, 92%, 95%, 97%, or 98%, 99% identical) to a nucleotide sequence as set forth in Table 1, Table 2, or Table 3. In certain embodiments, isolated nucleic acid molecules that encode at least one of the heavy chain (HC) sequences of the anti-TMPRSS6 antibodies disclosed herein may comprise a nucleotide sequence selected from at least one of: SEQ ID NO: 5 or a sequence substantially identical to SEQ ID NO: 5; SEQ ID NO: 15 or a sequence substantially identical to SEQ ID NO: 15; SEQ ID NO. 25 or a sequence substantially identical to SEQ ID NO: 25: SEQ ID NO: 35 or a sequence substantially identical to SEQ ID NO: 35; SEQ ID NO: 45 or a sequence substantially identical to SEQ ID NO: 45; SEQ ID NO: 55 or a sequence substantially identical to SEQ ID NO: 55; SEQ ID NO: 62 or a sequence substantially identical to SEQ ID NO: 62; SEQ ID NO: 66 or a sequence substantially identical to SEQ ID NO: 66; SEQ ID NO: 70 or a sequence substantially identical to SEQ ID NO: 70; SEQ ID NO: 74 or a sequence substantially identical to SEQ ID NO: 74; SEQ ID NO: 78 or a sequence substantially identical to SEQ ID NO: 78, or SEQ ID NO: 82 or a sequence substantially identical to SEQ ID NO: 82. In certain embodiments, isolated nucleic acid molecules that encode at least one of the light chain (LC) sequences of the anti-TMPRSS6 antibodies or antigen-binding fragments thereof disclosed herein may comprise a nucleotide sequence selected from at least one of: SEQ ID NO: 10 or a sequence substantially identical to SEQ ID NO: 10; SEQ ID NO: 20 or a sequence substantially identical to SEQ ID NO: 20; or SEQ ID NO: 30 or a sequence substantially identical to SEQ ID NO: 30; SEQ ID NO: 40 or a sequence substantially identical to SEQ ID NO: 40; SEQ ID NO: 50 or a sequence substantially identical to SEQ ID NO: 50; SEQ ID NO: 60 or a sequence substantially identical to SEQ ID NO: 60; SEQ ID NO: 64 or a sequence substantially identical to SEQ ID NO: 64; SEQ ID NO: 68 or a sequence substantially identical to SEQ ID NO: 68; SEQ ID NO: 72 or a sequence substantially identical to SEQ ID NO: 72; SEQ ID NO: 76 or a sequence substantially identical to SEQ ID NO: 76; SEQ ID NO: 80 or a sequence substantially identical to SEQ ID NO: 80, or SEQ ID NO: 84 or a sequence substantially identical to SEQ ID NO: 84.
In another aspect, vector is provided comprising one or more nucleic acid molecules that encode at least one amino acid sequence of the anti-TMPRSS6 antibodies disclosed herein. In certain embodiments, a vector is provided comprising one or more nucleic acid molecules that encode at least one of the heavy chain (HC) or light chain (LC) sequences of the anti-TMPRSS6 antibodies disclosed herein. In certain embodiments, a vector is provided comprising nucleic acid molecules that encode at least a portion of at least one of the amino acid sequences as set forth in Table 1, Table 2, or Table 3, or at least a portion of an amino acid sequence substantially identical to an amino acid sequence as set forth in Table 1, Table 2, or Table 3. In certain embodiments, a vector is provided comprising nucleic acid molecules that encode at least a portion of at least one of the HC or LC sequences as set forth in Table 1, Table 2, or Table 3, or at least a portion of an amino acid sequence substantially identical to at least one of the HC or LC sequences as set forth in Table 1, Table 2, or Table 3.
In another aspect, at least one host cell is provided containing a vector comprising one or more nucleic acid molecules that encode amino acid sequences of the anti-TMPRSS6 antibodies disclosed herein. In certain embodiments, a host cell is provided containing a vector comprising nucleic acid molecules that encode at least a portion of at least one of the HC or LC sequences as set forth in Table 1, Table 2, or Table 3, or at least a portion of an amino acid sequence substantially identical to at least one of the HC or LC sequences as set forth in Table 1, Table 2, or Table 3. In certain embodiments, at least one host cell is capable of supporting vector expression and recombinant production of anti-TMPRSS6 antibodies or antigen-binding fragments thereof encoded by the vector. In certain embodiments, at least one host cell is capable of supporting vector expression and recombinant production of anti-TMPRSS6 antibodies or antigen-binding fragments thereof encoded by a vector comprising nucleic acid molecules that encode at least a portion of at least one of the HC or LC sequences as set forth in Table 1, Table 2, or Table 3, or at least a portion of an amino acid sequence substantially identical to at least one of the HC or LC sequences as set forth in Table 1, Table 2, or Table 3. In certain embodiments, host cells are transiently transfected with a vector comprising one or more nucleic acid molecules that encode amino acid sequences of the anti-TMPRSS6 antibodies or antigen-binding fragments thereof disclosed herein, wherein the host cells are capable of supporting vector expression and recombinant production of anti-TMPRSS6 antibodies or antigen-binding fragments thereof encoded by the vector.
In some aspects, the present disclosure provides a method for treating polycythemia vera (PV) in a subject. In some embodiments, the PV is associated with overactivation of JAK2/STAT5 pathway. In some embodiments, the method comprises administering to the subject an effective amount of an anti-TMPRSS6 antibody. In some embodiments, the antibody comprises a heavy chain complementarity-determining region 1 (HC CDR1) comprising the amino acid sequence of SEQ ID NO: 52, a HC CDR2 comprising the amino acid sequence of SEQ ID NO: 53, a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 54, a light chain complementarity-determining region 1 (LC CDR1) comprising the amino acid sequence of SEQ ID NO: 57, a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 58, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO: 59. In some embodiments, the antibody comprises a HC CDR1 comprising the amino acid sequence of SEQ ID NO: 2, a HC CDR2 comprising the amino acid sequence of SEQ ID NO: 3, a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 4, a LC CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the antibody comprises a HC CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a HC CDR2 comprising the amino acid sequence of SEQ ID NO: 13, a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 14, a LC CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antibody comprises a HC CDR1 comprising the amino acid sequence of SEQ ID NO: 22, a HC CDR2 comprising the amino acid sequence of SEQ ID NO: 23, a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 24, a LC CDR1 comprising the amino acid sequence of SEQ ID NO: 27, a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 28, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the antibody comprises a HC CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a HC CDR2 comprising the amino acid sequence of SEQ ID NO: 33, a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 34, a LC CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO: 39. In some embodiments, the antibody comprises a HC CDR1 comprising the amino acid sequence of SEQ ID NO: 42, a HC CDR2 comprising the amino acid sequence of SEQ ID NO: 43, a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 44, a LC CDR1 comprising the amino acid sequence of SEQ ID NO: 47, a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 48, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO: 49.
In some aspects, the present disclosure provides a method for treating polycythemia vera (PV) in a subject. In some embodiments, the subject has bone marrow that comprises cells with JAK2/STAT5 overactivation. In some embodiments, the method comprises administering the subject an effective amount of anti-TMPRSS6 antibody. In some embodiments, the antibody comprises a heavy chain complementarity-determining region 1 (HC CDR1) comprising the amino acid sequence of SEQ ID NO: 52, a HC CDR2 comprising the amino acid sequence of SEQ ID NO: 53, a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 54, a light chain complementarity-determining region 1 (LC CDR1) comprising the amino acid sequence of SEQ ID NO: 57, a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 58, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO: 59. In some embodiments, the antibody comprises a HC CDR1 comprising the amino acid sequence of SEQ ID NO: 2, a HC CDR2 comprising the amino acid sequence of SEQ ID NO: 3, a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 4, a LC CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the antibody comprises a HC CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a HC CDR2 comprising the amino acid sequence of SEQ ID NO: 13, a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 14, a LC CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antibody comprises a HC CDR1 comprising the amino acid sequence of SEQ ID NO: 22, a HC CDR2 comprising the amino acid sequence of SEQ ID NO: 23, a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 24, a LC CDR1 comprising the amino acid sequence of SEQ ID NO: 27, a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 28, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the antibody comprises a HC CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a HC CDR2 comprising the amino acid sequence of SEQ ID NO: 33, a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 34, a LC CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO: 39. In some embodiments, the antibody comprises a HC CDR1 comprising the amino acid sequence of SEQ ID NO: 42, a HC CDR2 comprising the amino acid sequence of SEQ ID NO: 43, a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 44, a LC CDR1 comprising the amino acid sequence of SEQ ID NO: 47, a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 48, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO: 49.
In some embodiments, the subject has a mutation that leads to JAK2/STAT5 overactivation.
In some embodiments, the subject has a JAK2 mutation.
In some embodiments, the JAK2 mutation is a JAK2 gene exon 14 mutation. In some embodiments, the JAK2 exon 14 mutation is V617F, H606Q, H608Y, L611V, L611S, V617I, C618F, C618R or absent of exon 14. In some embodiments, the JAK2 exon 14 mutation is V617F. In some embodiments, the subject is a homozygous of JAK2 V617F mutation.
In some embodiments, the JAK2 mutation is a JAK2 gene exon 12 mutation. In some embodiments, the JAK2 mutation is F537-K539delinsL, N542-E543del mutation, H538QK539L, V536-1546 dup11, V536-F547 dup, F537-1546dup10F547L, F537IK539I, H538-K539delinsL, H538-K539del, H538DK539LI540S, H538G, K539L, K539E, I540-E543delinsMK, 1540-E542delinsS, R541-E543delinsK, N542-E543del, D544-L545del, or 547insLI540-F547dup8.
In some embodiments, the subject has a JAK2 gene exon 15 mutation. In some embodiments, the JAK2 gene exon 15 mutation is L642P, or I645V.
In some embodiments, the subject has a non-Jak2 mutation. In some embodiments, the subject has a mutation in SRSF2 gene, SF3B1 gene, U2AF1 gene, U2AF1 gene, ZRSR2 gene, TET2 gene, DNMT3a gene, IDH1/IDH2 gene, ASXL1 gene, EZH2 gene, LNK/SH2B3 gene, NF-E2 gene, NF1 gene, CBL gene, FLT3 gene, ERBB gene, PPMID gene, TR53 gene, RUNX1 gene, CUX1 gene, ETV6 gene, CALR gene, MPL gene, let-7a gene, miR-26b gene, miR-27b gene, miR-28 gene, miR-30b gene, miR-30c gene, miR-125-5p gene, miR-125b-5p gene, miR-143 gene, miR-145 gene, miR-150 gene, miR-182 gene, miR-223 gene, miR-342 gene, or miR-451 gene.
In some embodiments, the mutation is an acquired mutation, a familial mutation, or a congenital mutation.
In some embodiments, the subject comprises hematopoietic progenitor cells that comprises the one or more of the mutations. In some embodiments, the one or more mutation occurs in CD34+CD38− hematopoietic progenitor cells. In some embodiments, the one or more mutation occurs in myeloid progenitor cells. In some embodiments, the one or more mutation occurs in Megakaryocytic-erythroid progenitor cells. In some embodiments, the subject presents with a phenotypic profile of PV prior to the administration.
In some embodiments, the subject has increased hematocrit (HCT) relative to a subject that does not have PV. In some embodiments, the subject has splenomegaly prior to the administration. In some embodiments, the subject has erythrocytosis prior to the administration. In some embodiments, the subject has leukocytosis prior to the administration. In some embodiments, the subject has thrombocytosis prior to the administration. In some embodiments, the subject has increased hemoglobin relative to a subject that does not have PV prior to the administration. In some embodiments, the subject has increased red cell distribution width (RDW) relative to a subject that does not PV prior to the administration.
In some embodiments, the administration of the antibody increases serum hepcidin. In some embodiments, administration of the antibody reduces liver iron. In some embodiments, the administration of the antibody reduces HCT. In some embodiments, the administration of the antibody reduces red blood cell count. In some embodiments, the administration of the antibody reduces red cell distribution width (RDW). In some embodiments, the administration of the antibody reduces serum iron. In some embodiments, the administration of the antibody reduces leukocytosis. In some embodiments, the administration of the antibody reduces early erythroid progenitor cells. In some embodiments, the administration of the antibody reduces plasma hemoglobin level. In some embodiments, the administration of the antibody reduces mean corpuscular volume (MCV). In some embodiments, the administration of the antibody reduces the frequency of thrombosis events (TEs). In some embodiments, the administration of the antibody reduces frequency of phlebotomy. In some embodiments, the administration of the antibody reduces frequency of cytoreductive therapy. In some embodiments, the antibody treats a subject in need thereof that is refractory to phlebotomy and/or cytoreductive therapy. In some embodiments, the administration of the antibody results in reduction of symptoms as described by Myeloproliferative Neoplasm Symptom Assessment Form (MPN-SAF).
In some embodiments, the subject is administered an antibody comprising a heavy chain complementarity-determining region 1 (HC CDR1) comprising the amino acid sequence of SEQ ID NO: 52, a HC CDR2 comprising the amino acid sequence of SEQ ID NO: 53, a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 54, a light chain complementarity-determining region 1 (LC CDR1) comprising the amino acid sequence of SEQ ID NO: 57, a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 58, and a LC CDR3 comprising the amino acid sequence of SEQ ID NO: 59.
In some embodiments, the subject is administered an antibody comprising a heavy chain variable region (VH) comprising the amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 51, and a light chain variable region (VL) comprising the amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 56. In some embodiments, the subject is administered an antibody comprising a VH comprising the amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 1, and VL comprising the amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the subject is administered an antibody comprising a VH comprising the amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 11, and VL comprising the amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the subject is administered an antibody comprising a VH comprising the amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 21, and VL comprising the amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 26. In some embodiments, the subject is administered an antibody comprising a VH comprising the amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 31, and VL comprising the amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 36. In some embodiments, the subject is administered an antibody comprising a VH comprising the amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 41, and VL comprising the amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 46. In some embodiments, the sequence variation occurs in the framework region of the VH and//or framework region of the VL, of an anti-TMPRSS6 antibody described herein.
In some embodiments, the subject is administered an antibody comprising a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 51, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 56.
In some embodiments, the subject is administered an antibody comprising a heavy chain comprising the amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 81, and a light chain comprising the amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 83. In some embodiments, the subject is administered an antibody comprising a heavy chain comprising the amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 61, and a light chain comprising the amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 63. In some embodiments, the subject is administered an antibody comprising a heavy chain comprising the amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 65, and a light chain comprising the amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 67. In some embodiments, the subject is administered an antibody comprising a heavy chain comprising the amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 69, and a light chain comprising the amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 71. In some embodiments, the subject is administered an antibody comprising a heavy chain comprising the amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 73, and a light chain comprising the amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 75. In some embodiments, the subject is administered an antibody comprising a heavy chain comprising the amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 77, and a light chain comprising the amino acid sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to the amino acid sequence of SEQ ID NO: 79. In some embodiments, the sequence variation occurs in the framework region of the VH, framework region of the VL, heavy chain constant region, and/or light chain constant region of an anti-TMPRSS6 antibody described herein.
In some embodiments, the subject is administered an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 81, and a light chain comprising the amino acid sequence of SEQ ID NO: 83.
In some embodiments, the antibody cross-reacts with at least one non-human TMPRSS6. In some embodiments, the non-human TMPRSS6 is a mouse TMPRSS6 or a non-human primate TMPRSS6. In some embodiments, the antibody specifically binds to human TMPRSS6. In some embodiments, the antibody does not specifically bind to human matriptase-1 or human matriptase-3.
In some embodiments, the subject receives one or more additional therapeutic for treating PV. In some embodiments, the additional therapeutic for treating PV comprising interferon (e.g., ropeginterferon a-2b-njft (Besremi), pegylated interferon), JAK2 inhibitor (e.g., ruxolitinib, XL019, fedratinib (SAR302503), momelotinib), JAK1 inhibitor (e.g., itacitinib), hepcidin memetic (e.g., rusfertide (PTG-300)), lysine specific demethylase inhibitor (e.g., Bomedemstat (IMG-7298), TMPRSS6 antagonist (e.g., Sapablursen (ISIS 702843), SLN124), anti-TfR1 antibody (e.g., PPMX-T003), MDM2 inhibitor (e.g., Idasanutlin (RG7388), KRT-232), tyrosine kinase inhibitor (e.g., Dasatinib, Erlotinib, Gleevec, lestaurtinib (CEP-701)), HDAC inhibitor (e.g., Givinostat (ITF2357), MK-0683), PI3K inhibitor (e.g., Umbralisib (TGR-1202), telomerase inhibitor (e.g., Imetelstat), phlebotomy, low-dose aspirin, or hydroxyurea.
The invention relates to novel antibodies and antigen-binding fragments thereof that bind TMPRSS6, and methods of making and using the same.
Scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art, unless otherwise defined. Use of singular terms (“a” or “an” or “the” or other use of a term in the singular) include plural reference, and plural terms shall include the singular, unless the context clearly dictates otherwise. Thus, for example, reference to “an antibody” includes “one or more” antibodies or a “plurality” of such antibodies. All publications mentioned herein are hereby incorporated by reference in their entirety.
Generally, nomenclature and techniques of molecular biology, microbiology, cell and tissue culture, protein and nucleotide chemistry, and recombinant DNA techniques available to one of skill of the art can be employed for the antibodies, antigen-binding fragments, compositions, and methods disclosed herein. Techniques and procedures described herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references, inter alia, Sambrook et al. (1989) MOLECULAR CLONING: A LABORATORY MANUAL (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and Ausubel et al. (1994) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Volumes I-III (John Wiley & Sons, N.Y.). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein, unless otherwise specified herein. Techniques and methods for pharmaceutical preparation and formulation, and treatment of subjects, are described herein using conventional nomenclature.
“Antibody” refers in the broadest sense to a polypeptide or combination of polypeptides that recognizes and binds to an antigen through one or more immunoglobulin variable regions, where the immunoglobulin variable regions may be naturally occurring or non-naturally occurring, e.g., as a result of engineering, chimerization, humanization, optimization, CDR-grafting, or affinity maturation.
An “antibody” as disclosed herein can be a whole (intact, full length) antibody, a single chain antibody, or an antigen binding fragment with one or two chains, and can be naturally occurring and non-naturally occurring. An antibody comprises at least sufficient complementarity determining regions (CDR), interspersed with framework regions (FR), for the antibody to recognize and bind to an antigen. An anti-TMPRSS6 antibody disclosed herein may be, but is not limited to, at least one of a monoclonal antibody, a recombinant monoclonal antibody, a polyclonal antibody, a humanized antibody, a chimeric antibody, a single chain antibody, a Fab fragment, a single-chain variable fragment (scFv), an aptamer, a single-domain antibody (VHH or nanobody), a recombinant antibody, a modified antibody having peptide/other moieties attached to antibody and/or additional amino acids added the N- or C-terminus, or other TMPRSS6-binding fragment or variant. Whole antibody, full length antibody, intact antibody, naturally occurring antibody, or equivalent terms are understood to refer to a polypeptide, in particular a glycoprotein, comprising at least two heavy chains (HCs) and two light chains (LCs) interconnected by disulfide bonds. Each HC is comprised of a heavy chain variable region (VH) and an HC constant region (CH), and each light chain is comprised of a light chain variable region (VL) and an LC constant region (CL). The HC and LC variable regions, VH and VL, include a binding domain that interacts with an antigen. The VH and VL regions can be further subdivided into CDR regions characterized by hypervariability, interspersed with FR regions that are typically more conserved. Each VH and VL is typically composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system and the classical complement system. Typically, an antibody comprises at least heavy chain (HC) CDR1, CDR2, and CDR3 and light chain (LC) CDR1, CDR2, and CDR3 sequences, where any one of these sequences may be naturally or non-naturally occurring. An antibody may comprise fewer CDR sequences, as long as the antibody can recognize and bind an antigen.
An anti-TMPRSS6 antibody disclosed herein may be a variant comprising at least one altered CDR or framework sequence, wherein CDR and/or framework sequences may by optimized by mutating a nucleic acid molecule encoding such framework sequence. Variants may be constructed with HC and LC portions derived independently from different sources. Techniques for generating variants include but are not limited to conservative amino acid substitution, computer modeling, screening candidate polypeptides alone or in combinations, and codon optimization, and it is understood that a skilled person is capable of generating antibody variants as may be needed. An anti-TMPRSS6 antibody disclosed herein may be a fragment. Antigen binding functions of an antibody can be performed by fragments such as: a Fab fragment; a monovalent fragment consisting of the VL, VH, CL and CHI domains; a F (ab) 2 fragment; a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; an Fd fragment consisting of the VH and CHI domains; a single-chain variable fragment (scFv) consisting of the VL and VH domains of a single arm of an antibody; a single domain antibody (dAb) fragment which consists of a VH domain; and an isolated CDR (VHH, nanobody), or an aptamer. Antigen binding portions can be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136). Antigen binding portions of antibodies can be grafted into scaffolds based on polypeptides to form monobodies (see, e.g., U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies).
The term antibody encompasses various broad classes of polypeptides that can be distinguished biochemically. The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. Those skilled in the art understand that there are five major classes of antibodies, viz., IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, each of which is well characterized and known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernable and within the scope of the instant disclosure. While all immunoglobulin classes are within the scope of the present disclosure, the present disclosure will be directed largely to the IgG class of immunoglobulin molecules.
The term “chimeric” antibody refers to an antibody in which a portion of the heavy chain (HC) and/or light chain (LC) involved in forming the immunoreactive site is derived from a particular source or species, while the remainder of the HC and/or LC is derived from a different source or species. In certain embodiments the target binding region or site will be from a non-human source (e.g., mouse or non-human primate) and the constant region is human.
As used herein, the phrase “humanized antibody” refers to an antibody or antibody variant derived from a non-human antibody, typically a mouse monoclonal antibody, where CDRs from the parental, non-human antibody are grafted (fused) in a framework comprising variable regions derived from a human immunoglobulin framework, in particular an acceptor human framework or a human consensus framework. Techniques and principles for designing, making, and testing humanized antibodies are known (Jones P T, Dear P H, Foote J, Neuberger M S, Winter G. Replacing the complementarity-determining regions in a human antibody with those from a mouse. Nature. 1986 May 29-Jun. 4; 321 (6069): 522-5; Almagro J C, Fransson J. Humanization of antibodies. Front Biosci. 2008 Jan. 1; 13:1619-33). It is understood that changes can be made to an acceptor framework at multiple locations in order to develop a humanized antibody having improved features according to the desired use, e.g., high affinity for target, low clearance, low toxicity, etc. An anti-TMPRSS6 antibody disclosed herein may be a humanized variant.
“Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, binding affinity as used herein refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). Affinity can be measured by common methods known in the art, including those described herein. The calculated concentration at which approximately 50% of maximal binding (the calculated EC50) can be used as an estimate of affinity. The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd or KD, representing koff/kon measured for the interaction).
A “subject” is a mammal, where mammals include but are not limited to primates (e.g., humans and non-human primates such as monkeys), domesticated animals (e.g., cows, sheep, cats, dogs, pigs, llamas, and horses), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the subject is a human. The phrases “to a subject in need thereof” or “to a patient in need thereof” or “to a patient in need of treatment” or “a subject in need of treatment” may include subjects that would benefit from administration of the anti-TMPRSS6 antibodies disclosed herein, for treatment of an iron overload disorder. It is understood that administration of anti-TMPRSS6 antibodies encompasses administration to “a subject in need thereof” can be interpreted as referring to a subject known or suspected to have an iron overload disorder, in particular a β-thalassemia, based on indicators such as symptoms, family history, or genotype. It is further understood that anti-TMPRSS6 antibodies can be administered to a subject that is not known or suspected to have a disorder of iron metabolism, for purposes that may include but are not limited to, preventative or prophylactic purposes, for screening, for diagnostics, for research purposes, or to achieve results distinct from treating a disorder.
An “effective amount” of an anti-TMPRSS6 antibody, e.g., in a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. It is understood that “effective amount” is intended to refer to the amount of an anti-TMPRSS6 antibody or a pharmaceutical composition comprising an anti-TMPRSS6 antibody that will elicit the biological response of, or desired therapeutic effect on, a cell, a tissue, a system, a non-human animal subject, a non-human mammal subject, or a human subject that is being measured. The terms “therapeutically effective amount”, “pharmacologically effective amount”, and “physiologically effective amount” are used interchangeably to refer to the amount of an anti-TMPRSS6 antibody that is needed to provide a threshold level of active agents in the bloodstream or in the target tissue. The precise amount will depend upon numerous factors, e.g., the particular anti-TMPRSS6 antibody (active agent), the components and physical characteristics of the composition, intended population of subjects/patients to be treated, considerations such as the disease state, age, sex, and weight of a subject, and the like, and can readily be determined by one skilled in the art, based upon the information provided herein or otherwise available in the relevant literature. The terms, “improve”, “increase” or “reduce”, as used in this context, indicate values or parameters relative to a baseline measurement, such as a measurement in the same subject prior to initiation of the treatment described herein, or a measurement in a control individual (or multiple control individuals) in the absence of the treatment described herein.
The term “pharmaceutical composition” or “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, in particular an anti-TMPRSS6 antibody. It is understood that a pharmaceutical composition may contain more than one active ingredient, e.g., more than one anti-TMPRSS6 antibody, or a combination of an anti-TMPRSS6 antibody with another active ingredient that acts on a different target, where such combinations can be but are not limited to, a combination of an antiTMPRSS6 antibody with another active ingredient having a desired effect on hematopoietic processes, in particular erythropoiesis, a combination of an anti-TMPRSS6 antibody with gene therapy agents such as agents to carry out gene therapy targeting the HBB gene, or a combination of an anti-TMPRSS6 antibody with Fc-fusion proteins that target TGF superfamily ligands to stimulate erythropoiesis. A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. It is understood that a pharmaceutically acceptable carrier can be, but is not limited to, a buffer, excipient, stabilizer, an adjuvant, or preservative.
The term “Janus kinase 2/signal transducer and activator of transcription protein 5 (JAK2/STAT5) pathway” or “JAK2/STAT5 signaling”, as used herein, refers to a cell signaling pathway (e.g., involved in processes such as immunity, cell division, cell death, and tumour formation) mediated by Janus kinases 2 (JAK2), signal transducer and activator of transcription protein 5 (STAT5)), receptors associated with JAK2, and other regulator proteins. JAK2 is a member of the family of Janus kinases, which are non-receptor protein tyrosine kinases. JAKs (e.g., JAK2) associates with the cytoplasmic domain of several cytokine receptors (e.g., receptors lacking intrinsic kinase activity, but are not limited to interferon receptors, the GM-CSF receptor family receptors (e.g., IL-3R, IL-5R and GM-CSF-R), the gp130 receptor family receptors (e.g., IL-6R), and the single chain receptors (e.g. Epo-R, Tpo-R, GH-R, PRL-R)). JAK2 is activated through tyrosine-phosphorylation of the cytoplasmic domains of cytokine receptors, upon cytokine binding. JAK2 activation promotes recruitment to the receptor complex of the transcription factors signal transducer and activator of transcription (e.g., STAT5). (see, e.g., Levine et al., Role of JAK2 in the pathogenesis and therapy of myeloproliferative disorders. Nat Rev Cancer. 2007; 7:673-683). JAK2 further phosphorylates STAT (e.g., STAT5), and JAK2-mediated STAT phosphorylation leads to the formation of stable homodimers or heterodimers of STAT (e.g., STAT5 homodimers). Phosphorylated STAT dimer (e.g., STAT5 homodimer) translocate to the nucleus and binds promoters of the downstream genes to initiate transcription of these genes.
In some embodiments, JAKs possess a pseudokinase domain (i.e., JH2 domain), which is upstream of the C-terminal tyrosine kinase domain (i.e., JH1 domain). In some embodiments, the pseudokinase domain of JAKs is crucial for maintaining a low basal (e.g., absence of cytokine) level of tyrosine kinase activity (see, e.g., Hubbard, Mechanistic Insights into Regulation of JAK2 Tyrosine Kinase, Frontiers in Endocrinology, January 2018, Volume 8, Article 361).
The term “Overactivation of JAK2/STAT5 pathway”, or “JAK2/STAT5 overactivation”, as used herein, refers to aberrant activation (e.g., increased activation, constitutive activation) of JAK2/STAT5 pathway compared to a physiologically normal level of activation. In some embodiments, overactivation of JAK2/STAT5 can be determined by comparing the JAK2/STAT5 activity measured by any suitable method to JAK2/STAT5 activity in a normal subject. For example, in some embodiments, overactivation of JAK2/STAT5 can be determined by comparing the JAK2/STAT5 activity in cells of a subject having a mutant JAK2 allele to JAK2/STAT5 activity in a normal subject having only wild-type JAK2 alleles, for purposes of determining whether the JAK2 mutation is associated with over activation. In some embodiments, similar analyses may be performed to determine the effects of other genetic alterations (e.g., in STAT5 or other genes) on JAK2/STAT5 signaling. Activity of JAK2/STAT5 pathway can be measured by any suitable methods known in the art, for example, kinase activity assays (e.g., JAK2 assay kit Catalog #79520 by BPS Bioscience; PathHunter® eXpress EpoR-JAK2 Functional Assay Catalog No. 93-0900E3CP by Eurofins; HTScan® Jak2 Kinase Assay Kit, Catalog No. 7752 by Cell Signaling Technology, etc.); western blot (e.g., western blot for phosphorylated JAK2, STAT5, and/or downstream proteins); RT-PCR (e.g., RT-PCR for expression level of the downstream genes).
In some embodiments, a subject is a subject having polycythemia vera (PV). In some embodiments, the PV is associated with overactivation of JAK2/STAT5 overactivation. In some embodiments, a subject having PV has JAK2/STAT5 activity that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% higher than that of a subject who does not have PV. In some embodiments, a subject is a subject having polycythemia vera (PV). In some embodiments, the PV is associated with overactivation of JAK2/STAT5 overactivation. In some embodiments, a subject having PV has JAK2/STAT5 activity that is at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 50 times, at least 75 times, at least 100 times or higher than that of a subject who does not have PV. In some embodiments, a subject having PV has JAK2/STAT5 activity that is up to 1.5 times, up to 2 times, up to 3 times, up to 4 times, up to 5 times, up to 6 times, up to 7 times, up to 8 times, up to 9 times, up to 10 times, up to 15 times, up to 20 times, up to 25 times, up to 50 times, up to 75 times, up to 100 times more than that of a subject who does not have PV. In some embodiments, a subject having PV has JAK2/STAT5 activity that is between 10% and 100 times, between 20% and 100 times, between 30% and 100 times, between 40% and 100 times, between 50% and 100 times, between 60% and 100 times, between 70% and 100 times, between 80% and 100 times, between 90% and 100 times, between 1 and 100 times, between 5 and 100 times, between 10 and 100 times, between 20 and 100 times, between 25 and 100 times, between 50 and 100 times, between 75 and 100 times, between 10% and 90 times, between 20% and 90 times, between 30% and 90 times, between 40% and 90 times, between 50% and 90 times, between 60% and 90 times, between 70% and 90 times, between 80% and 90 times, between 90% and 90 times, between 1 and 90 times, between 5 and 90 times, between 10 and 90 times, between 20 and 90 times, between 25 and 90 times, between 50 and 90 times, between 75 and 90 times, between 10% and 80 times, between 20% and 80 times, between 30% and 80 times, between 40% and 80 times, between 50% and 80 times, between 60% and 80 times, between 70% and 80 times, between 80% and 80 times, between 90% and 80 times, between 1 and 80 times, between 5 and 80 times, between 10 and 80 times, between 20 and 80 times, between 25 and 80 times, between 50 and 80 times, between 75 and 80 times, between 10% and 50 times, between 20% and 50 times, between 30% and 50 times, between 40% and 50 times, between 50% and 50 times, between 60% and 50 times, between 70% and 50 times, between 80% and 50 times, between 90% and 50 times, between 1 and 50 times, between 5 and 50 times, between 10 and 50 times, between 20 and 50 times, between 25 and 50 times, between 10% and 25 times, between 20% and 25 times, between 30% and 25 times, between 40% and 25 times, between 50% and 25 times, between 60% and 25 times, between 70% and 25 times, between 80% and 25 times, between 90% and 25 times, between 1 and 25 times, between 5 and 25 times, between 10 and 25 times, between 20 and 25 times, between 2 and 25 times, between 12 and 25 times, between 18 and 25 times, between 10% and 10 times, between 20% and 10 times, between 30% and 10 times, between 40% and 10 times, between 50% and 10 times, between 60% and 10 times, between 70% and 10 times, between 80% and 10 times, between 90% and 10 times, between 1 and 10 times, between 5 and 10 times, between 2 and 10 times, between 3 and 10 times, between 4 and 10 times, between 5 and 10 times, between 6 and 10 times, between 7 and 10 times, between 8 and 10 times, between 9 and 10 times, between 10% and 5 times, between 20% and 5 times, between 30% and 5 times, between 40% and 5 times, between 50% and 5 times, between 60% and 5 times, between 70% and 5 times, between 80% and 5 times, between 90% and 5 times, between 1 and 5 times, between 2 and 5 times, between 3 and 5 times, between 4 and 5 times, between 10% and 2 times, between 20% and 2 times, between 30% and 2 times, between 40% and 2 times, between 50% and 2 times, between 60% and 2 times, between 70% and 2 times, between 80% and 2 times, between 90% and 2 times, or between 1 and 2 times more than that of a subject who does not have PV.
The term “treat” or “treating” or similar terms as used herein, can refer to an outcome that is deemed beneficial for a particular subject in a defined set of circumstances. Treating a disorder of iron metabolism may refer non-exclusively to any of reducing, ameliorating, slowing, interrupting, arresting, alleviating, stopping, or reversing the progression or severity of an existing symptom, disorder, condition, or disease, and may further encompass prevention or delay of the onset of one or more symptoms of an iron overload disorder, and/or lessening of the severity or frequency of one or more symptoms of an iron overload disorder. The terms “treating” or “method of treating” or equivalents can encompass one or more uses of anti-TMPRSS6 antibodies disclosed herein, including but not limited to therapeutic, prophylactic, preventive, diagnostic, imaging, and screening uses.
The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating a nucleic acid to which the vector sequence is linked, in a host cell in which the vector is introduced. Vectors capable of directing the expression of nucleic acids to which they are operatively linked are referred to herein as “expression vectors.”
Antibodies and antigen-binding fragments are provided that are capable of binding TMPRSS6 on the surface of a cell and modulating the activity of at least one component involved in iron metabolism, in particular at least one component involved in iron overload disorders associated with abnormal suppression of hepcidin expression. Anti-TMPRSS6 antibodies that are capable of binding TMPRSS6 on the surface of a cell and modulating the activity of at least one component involved in regulating hepcidin expression can be used in methods for treating iron overload disorders associated with abnormal suppression of hepcidin expression. Anti-TMPRSS6 antibodies that are capable of binding TMPRSS6 on the surface of a cell and modulating TMPRSS6 suppression of hepcidin expression can be used to therapeutically target TMPRSS6 in methods for treating iron overload disorders and/or other iron dysregulation disorders and/or associated with abnormal suppression of hepcidin expression.
Once antibodies or fragments specific for TMPRSS6, in particular human TMPRSS6 expressed on the surface of a cell, have been obtained, the desired biological activity of modulating the activity of at least one component involved in iron metabolism thereof can be tested by several methods known to the skilled person.
It is understood that “modulate” or “modulating” or similar terms as used herein can refer to one or more effects that can result when an anti-TMPRSS6 antibody disclosed herein binds its target. “Modulating” and its equivalents can refer to different modes of action and effects depending on the component under consideration, i.e., modulating can refer to neutralizing, reversing, inhibiting, blocking, reducing, antagonizing, or otherwise interfering with the activity of certain components involved in iron metabolism, while for other components involved in iron metabolism the term modulating can refer to increasing, enhancing, or having an agonist effect on these components.
It is understood that the term “component” can refer not only to target molecule TMPRSS6, but also to a downstream process or pathway involved in iron metabolism. Thus, a component within the meaning of a process or pathway can be, but is not limited to, regulation of hepcidin expression, TMPRSS6 suppression of hepcidin expression, the process of hepcidin expression, regulation of hepcidin levels, increasing hepcidin levels, the activity of the hepcidin promoter, or TMPRSS6 suppression of the BMP/SMAD pathway-induced expression of hepcidin, regulation of liver non-heme iron levels, one or more processes involved in splenomegaly, or one or more hematopoietic processes involved in regulation of red blood count (RBC), hematocrit (HCT), red cell distribution width (RDW), and erythropoiesis, in particular production of mature red cells.
Anti-TMPRSS6 antibodies as disclosed herein can be used to therapeutically target at least one component involved in iron metabolism, in particular at least one component involved in iron overload disorders. In certain embodiments, anti-TMPRSS6 antibodies as disclosed herein can be used to therapeutically target at least one component involved in regulating hepcidin expression, and modulate the activity of the component to achieve increased hepcidin expression. In certain embodiments, anti-TMPRSS6 antibodies as disclosed herein can be used to modulate the activity of the hepcidin promoter to achieve increased hepcidin expression. It is understood that anti-TMPRSS6 antibodies as disclosed herein can be used to therapeutically target TMPRSS6 and thereby modulate the downstream activity of other components of hepcidin expression, including but not limited to, regulation of liver non-heme iron levels, one or more processes involved in splenomegaly, or one or more hematopoietic processes involved in regulation of red blood count (RBC), hematocrit (HCT), red cell distribution width (RDW), and erythropoiesis, in particular production of mature red cells.
Using anti-TMPRSS6 antibodies as disclosed herein to therapeutically target at least one component involved in iron metabolism, allows precise modulation of the targeted component. It is understood that by using anti-TMPRSS6 antibodies as disclosed herein to precisely target TMPRSS6 and its downstream effects on at least one component involved in regulating hepcidin expression, it is possible to avoid undesirable effects, difficulties with delivery and/or effectiveness, and regulatory hurdles associated with other approaches to treating iron overload disorders that are currently in use or under development, e.g., blood transfusions that can further exacerbate iron overload, iron chelation with poor patient compliance, intrusive phlebotomy or splenectomy that only manage symptoms, gene therapy targeting the HBB gene with potential permanent pleiotropic effects in multiple systems, gene therapy and gene editing with unknown off-target effects, Fc-fusion proteins targeting TGF superfamily ligands to inhibit SMAD signaling that do not reduce the need for iron chelation therapy to manage iron overload, and other approaches that are difficult to control or deliver such as hepcidin mimetics, and antisense or iRNA drugs targeting TMPRSS6. It is understood that using anti-TMPRSS6 antibodies for precise therapeutic targeting does not exclude the possibility of using anti-TMPRSS6 antibodies in methods and compositions for combination treatments, e.g., in combination with another active ingredient that acts on a different target, in combination with an antibody that binds a different target, in combination with gene therapy agents and methods for targeting the HBB gene, or in combination with Fc-fusion proteins that target TGF superfamily ligands to stimulate erythropoiesis.
Anti-TMPRSS6 antibodies disclosed herein allow the development of treatments that can be tailored to each subject (e.g., dosage, frequency of administration), where they can be continued and discontinued with ease, and combined with other therapies. In certain strategic embodiments, anti-TMPRSS6 antibodies disclosed herein can be combined with other therapies that may address multiple therapeutic targets and/or address deficits or undesirable effects of one of the therapies in the combination therapy.
Non-limiting exemplary embodiments of anti-TMPRSS6 antibodies of the invention are presently disclosed, in particular in the Examples, Tables, and Figures.
a. Antibodies Capable of Binding TMPRSS6
As demonstrated in the Examples, a functional cascade can be used to identify and characterize anti-TMPRSS6 antibodies of the present invention, where a first step in the cascade involves screening for antibodies capable of binding to human TMPRSS6 on the surface of a cell expressing TMPRSS6 (Example 1,
In a third step of the functional cascade (
b. Humanized Variants
Humanized antibodies comprising CDRs derived from a non-human source grafted into a human-derived antibody framework are expected to be non-immunogenic when administered to a human subject. As demonstrated by exemplary embodiments disclosed in Example 2, humanized anti-TMPRSS6 antibody variants were successfully generated, tested, optimized, and selected. Multiple candidate HC and LC variants were developed wherein each HC or LC variant had the same CDR sequences but the variable region frameworks sequences could vary at over 90% of the framework positions, and these variants tested in different HC/LC combinations to identify combinations having desired features. After initial design and testing, variants that showed desired antigen binding affinity were selected for further evaluation and development, including but not limited to modification of some parental CDR sequences to avoid potential unwanted events such as aspartate isomerization, and modification of some constant regions (Fc) to achieve desired functions such as minimizing antibody-dependent cellular cytotoxicity (ADCC), to arrive at humanized variants hzMWTx-001 Var (SEQ ID NOs: 73 (HC) and 75 (LC)), hzMWTx-002 Var (SEQ ID NOs: 77 (HC) and 79 (LC)), and hzMWTx-003 Var (SEQ ID NOs: 81 (HC) and 83 (LC)).
c. Anti-TMPRSS6 Antibodies that Increase Hepcidin Promoter Activity
As disclosed herein, antibodies for use in treating iron overload disorders characterized by reduced hepcidin expression may modulate the activity of at least one component involved in hepcidin expression, where the component may be activity of the hepcidin promoter. As demonstrated by exemplary embodiments using an in vitro assay disclosed in Example 2, anti-TMPRSS6 antibodies MWTx-001, MWTx-002, MWTx-003, hzMWTx-001 Var, hzMWTx-002 Var, and hzMWTx-003 Var increased HAMP promoter activity in a dose-dependent manner (
d. Anti-TMPRSS6 Antibodies Having High Affinity for a Target in a Relevant Biological Context
Anti-TMPRSS6 antibodies showed high affinity for a biologically appropriate target, i.e., human TMPRSS6 expressed on the surface of a cell. As demonstrated by exemplary embodiments of affinity measurements using three different methods disclosed in Example 3 and
e. Anti-TMPRSS6 Antibodies Having Cross-Reactivity with Non-Human Targets
It is desirable for therapeutically useful antibodies or antibody fragments to have sufficient cross-reactivity with non-human targets (non-human homologues) from sources that would be relevant for further studies such as preclinical efficacy studies, animal models of disease, toxicology studies, etc., such that the antibodies or antibody fragments should recognize, e.g., a mouse homologue and/or a primate homologue such as from cynomolgus monkey. As demonstrated by exemplary embodiments disclosed in Example 4, MWTx-001, hzMWTx-001 Var, MWTx-003, and hzMWTx-003 Var showed detectable cross-reactivity with mouse TMPRSS6, while MWTx-001, MWTx-002, MWTx-003, hzMWTx-001 Var, hzMWTx-002 Var, and hzMWTx-003 Var showed detectable cross-reactivity with cynomolgus monkey TMPRSS6.
f. Anti-TMPRSS6 Antibodies Specifically Bind TMPRSS6 (Matriptase-2)
Antibodies with a high level of specific binding to a target protein and low cross-reactivity with homologous proteins in the same organism, are expected to have reduced or no off-target effects. Anti-TMPRSS6 antibodies provided here show high specificity for human TMPRSS6 (matriptase-2), making them suitable for use in targeted compositions and methods. As demonstrated by exemplary embodiments disclosed in Example 5 and illustrated in
g. Anti-TMPRSS6 Antibodies Having In Vivo Dose-Dependent Effects on Hormones and Symptoms Associated with Iron Overload Disorder
Antibodies that can increase the level of serum hepcidin, a hormone that controls iron absorption and mobilization from iron stores, are expected to reduce, ameliorate, or prevent symptoms of iron overload disorder, in particular to reduce, ameliorate, or prevent symptoms of elevated levels of serum iron. As demonstrated by exemplary embodiments shown in Example 6, administration of anti-TMPRSS6 monoclonal antibody MWTx-003 or humanized variant hzMWTx-003 Var to wildtype subjects, i.e., subject that is not known or suspected to have an iron overload, resulted in an increase in serum hepcidin levels (
h. Anti-TMPRSS6 Antibodies for Treating β-Thalassemia
Antibodies and antibody fragments that can relieve one or more symptoms of an iron overload disorder in vivo when administered to a subject exhibiting an animal model of the disease, i.e., a subject that is known or suspected to have an iron overload disorder, are expected to have therapeutic effectiveness for clinical use. As demonstrated by exemplary embodiments shown in Example 7 using the Th3/+ mouse model of β-thalassemia, administration of the anti-TMPRSS6 monoclonal antibody MWTx-003 resulted in multiple effects including but not limited to reducing liver non-heme iron, increasing serum hepcidin, increasing liver hepcidin RNA, reducing splenomegaly, increasing red blood count (RBC), increasing hematocrit (HCT), reducing red cell distribution width (RDW), and increased production of mature red cells (increased erythropoiesis), compared with isotype controls. Each of these effects can be understood as an amelioration of a symptom of the disorder. Symptoms of the disorder are manifested in multiple biological systems that include but are not limited to effects in the liver (effects on liver non-heme iron, liver hepcidin RNA), in the blood (effects on serum iron levels, circulating hormone levels in particular serum hepcidin levels, RBC, HCT, RDW), spleen size and function (splenomegaly), and erythropoiesis in multiple sites including but not limited to bone marrow and spleen (effects on abundance of different precursor cell types and abundance of mature red cells in erythropoietic sites). Administration of anti-TMPRSS6 antibodies ameliorated multiple symptoms throughout the disease model subject, shifting the measured symptom levels away from levels seen in isotype controls for the disease model (untreated disease) and towards the levels seen in wildtype littermates that represent normal levels in a genetically similar subject that is not known or suspected to have the disease. Without wishing to be bound by a theory or mechanism of action, it is understood that ineffective erythropoiesis is a driving force for abnormal hepcidin suppression leading to increased iron absorption and iron overload, such that a treatment that improves erythroblast differentiation and maturation into red cells should be therapeutically beneficial for treating an iron overload disorder. The present non-limiting exemplary embodiment discloses an anti-TMPRSS6 antibody therapy that increased erythroblast differentiation and maturation into red cells and also decreased iron loading.
i. Anti-TMPRSS6 Antibodies for Treating Polycythemia Vera (PV)
Accordingly, certain aspects of the disclosure provides methods and related compositions for treating conditions associated with dysregulated iron metabolism in subjects having JAK2/STAT5 overactivation related myeloproliferative neoplasms (MPN) (e.g., polycythemia vera). Aspects of the disclosure related to methods and compositions (e.g., anti-TMPRSS6 antibodies) useful for regulating iron metabolism for treating polycythemia vera in a subject having JAK2/STAT5 overactivation.
Polycythemia vera (PV) is a chronic myeloproliferative neoplasm. PV is characterized by erythrocytosis, bone marrow erythroid and megakaryocytic hyperplasia, fatigue, aquagenic pruritus, microvascular symptoms, and symptomatic splenomegaly. In some embodiments, overactivation of JAK2/STAT5 pathway leads to unregulated proliferation of a cell (e.g., hematopoietic progenitor cell), thus leading to erythrocytosis, leukocytosis, and thrombocytosis in subjects having PV. Complications of PV include increased risk of arterial and venous thrombosis and the potential for evolution to myelofibrosis (MF) and MPN-blast phase (see, e.g., Polycythemia vera: the natural history of 1213 patients followed for 20 years. Gruppo Italiano Studio Policitemia. Ann Intern Med. 1995; 123:656-64; Passamonti et al., Life expectancy and prognostic factors for survival in patients with polycythemia vera and essential thrombocythemia. Am J Med. 2004; 117:755-61; Stein et al., Polycythemia vera: an appraisal of the biology and management 10 years after the discovery of JAK2 V617F. J Clin Oncol. 2015; 33:3953-60).
In some embodiments, a subject having PV presents with a phenotypic profile of PV prior to treatment (e.g., treatment described herein). In some embodiments, a subject have erythrocytosis relative to a subject not having PV. Erythrocytosis refers to production pf excess red blood cells. Erythrocytosis can be evaluated by measuring hematocrit (HCT), hemoglobin, and bone marrow cell morphology in the subject (see, e.g., Mithoowani et al., Investigation and management of erythrocytosis, CMAJ. 2020 Aug. 10; 192 (32): E913-E918).
In some embodiments, a subject having PV-associated erythrocytosis has increased hematocrit (HCT) relative to a subject not having PV. Hematocrit is the percentage by volume of red cells in your blood. A normal range of HCT is between about 38% and 48% for men, and between about 35% and 45% for women. In some embodiments, a subject having PV have erythrocytosis and presents with an increased HCT level of higher than 48% in women and 52% in men. In some embodiments, a subject having PV have HCT level of higher than 48%, higher than 49%, higher than 50%, higher than 51%, higher than 52%, higher than 53%, higher than 54%, higher than 55%, higher than 56%, higher than 57%, higher than 58%, higher than 59%, higher than 60%, higher than 61%, higher than 62%, higher than 63%, higher than 64%, higher than 65%, higher than 66%, higher than 67%, higher than 68%, higher than 69%, higher than 70%, higher than 71%, higher than 72%, higher than 73%, higher than 74%, higher than 75%, or higher. In some embodiments, a subject having PV with high HCT levels are at a greater risk of developing thrombotic events (TEs). In some embodiments, increased TEs leads to more complications, such as cardiovascular complications, thereby increasing morbidity and mortality of PV. In some embodiments, cytoreductive therapy, phlebotomy and/or apheresis may be used to reduce excess red blood cells to reduce HCT. HCT can be measured by any suitable known methods in the art. Cytoreductive therapy refers to certain medications that are used to treat reduce levels of blood cells. Cytoreductive therapy includes interferon therapies, hydroxyurea, hydroxycarbamide, ruxolitinib, and/or anagrelide.
In some embodiments, a subject having PV-associated erythrocytosis has increased hemoglobin (Hb) level relative to a subject not having PV. A normal range of Hb is between about 13 g/dL and 17 g/dL for men, and between about 11 g/dL and 15 g/dL for women. In some embodiments, a subject having PV presents with an increased Hb level of higher than 16.5 g/dL in men and higher than 16 g/dL in women. In some embodiments, a subject having PV have Hb level of higher than 16 g/dL, higher than 16.2 g/dL, higher than 16.5 g/dL, higher than 16.8 g/dL, higher than 17 g/dL, higher than 17.2 g/dL, higher than 17.5 g/dL, higher than 17.8 g/dL, higher than 18 g/dL, higher than 18.2 g/dL, higher than 18.5 g/dL, higher than 18.8 g/dL, higher than 19 g/dL, higher than 19.2 g/dL, higher than 19.5 g/dL, higher than 19.8 g/dL, higher than 20 g/dL, or higher. Hemoglobin level can be measured by any suitable known methods in the art.
In some embodiments, a subject having PV-associated erythrocytosis shows hypercellularity with trilineage in bone marrow biopsy. In some embodiments, hypercellularity with trilineage in bone marrow presents as prominent erythroid, granulocytic and megakaryocytic proliferation with pleomorphic, mature megakaryocytes (subject (see, e.g., Mithoowani et al., Investigation and management of erythrocytosis, CMAJ. 2020 Aug. 10; 192 (32): E913-E918). Bone marrow cell morphology can be examined by any suitable methods known in the art, e.g., bone marrow biopsy.
In some embodiments, a subject having PV-associated erythrocytosis may have low serum erythropoietin (EPO) level (e.g., less than 3 mU/mL, less than 3 mU/mL, less than 2.9 mU/mL, less than 2.8 mU/mL, less than 2.7 mU/mL, less than 2.6 mU/mL, less than 2.5 mU/mL, less than 2.4 mU/mL, less than 2.3 mU/mL, less than 2.2 mU/mL, less than 2.1 mU/mL, or less. Serum EPO can be measured in any suitable know methods in the art.
In some embodiments, a subject have increase red cell distribution width (RDW) relative to a subject not having PV. A red cell distribution width (RDW) test measures the differences in the volume and size of your red blood cells (erythrocytes). In some embodiments, high RDW is associated with higher risk of thrombosis in PV subjects (see, e.g., Liu et al., RBC distribution width predicts thrombosis risk in polycythemia vera, Leukemia volume 36, pages 566-568 (2022).
In some embodiments, a subject have leukocytosis relative to a subject not having PV. Leukocytosis refers to a high white blood cell count than normal. In some embodiments, a subject having PV exhibits leukocytosis in the absence of other factors that may cause increased white blood cell count (e.g., infection, acute inflammation).
In some embodiments, a subject having PV has splenomegaly. In PV, the bone marrow produces too many red blood cells, white blood cells, and platelets, which may cause the spleen to get bigger. In some embodiments, splenomegaly indicates disease progression in PV (Lee, et al., Volumetric Splenomegaly in Patients With Polycythemia Vera, J Korean Med Sci. 2022 Mar. 21; 37 (11): e87).
In some embodiments, PV patients are iron deficient at presentation and/or during the course of their disease. However, the co-existence of iron deficiency and polycythemia presents a physiological disconnect. In some embodiments, a subject having PV exhibits hepcidin suppression (i.e., low hepcidin level) compared to subjects do not have PV). In some embodiments, hepcidin suppression in subject having PV results from increased erythropoietic activity. In some embodiments, increased erythropoiesis suppress hepcidin via the erythroid-secreted hormone erythroferrone (ERFE) (Kautz et al., Identification of erythroferrone as an erythroid regulator of iron metabolism. Nat Genet. 2014; 46 (7): 678-684). In some embodiments, hepcidin suppression in subject having PV results from iron deficiency. Under normal conditions, suppression of hepcidin results in iron mobilization from hepatocyte stores, export after recycling by splenic macrophages, and increased iron absorption by duodenal enterocytes, which would result in recovery from iron deficiency. However, in some embodiments, in PV subject (e.g., PV subject having one or more JAK2 mutations described herein), suppression of hepcidin led to greater iron deficiency (see, e.g., Ginzburg et al., Dysregulated iron metabolism in polycythemia vera: etiology and consequences, Leukemia volume 32, pages 2105-2116 (2018)). In some embodiments, other conditions in PV, such as inflammation, counters hepcidin suppression. Aberrant hepcidin expression combined with iron deficiency suggests that disordered iron metabolism is an important component of the pathobiology of PV. In some embodiments, a subject having PV has decreased serum hepcidin relative to a subject not having PV. In some embodiments, a subject having PV has decreased serum pro-hepcidin relative to a subject not having PV (Kwapisz et al., Decreased serum prohepcidin concentration in patients with polycythemia vera, J Zhejiang Univ Sci B. 2009 November; 10 (11): 791-795). Hepcidin or pro-hepcidin can be measured by suitable know method in the art, e.g., hepcidin prohormone ELISA, hepcidin ELISA, RT-PCR, etc. While the exact mechanism of hepcidin suppression and iron deficiency in PV patients remain elusive, in some embodiments, previous studies have shown that therapeutics that increases hepcidin level (e.g., ruxolitinib, hepcidin mimetics) in subjects having PV improves PV associated symptoms (e.g., reduced hematocrit (HCT), reduced splenomegaly) (see, e.g., Verstovsek et al., Markers of iron deficiency in patients with polycythemia vera receiving ruxolitinib or best available therapy. Leuk Res. 2017; 56:52-59. May; Casu et al., Minihepcidin peptides as disease modifiers in mice affected by β-thalassemia and polycythemia vera. Blood. 2016; 128:265-76).
In some embodiments, the development PV in a subject is associated with a mutation in a cell (e.g., a cell in the bone marrow such as hematopoietic progenitor cell). In some embodiments, a subject having PV has a mutation of the JAK2 gene (e.g., a JAK2 mutation that leads to JAK2/STAT5 overactivation) (see, e.g., Tefferi et al., Targeted Deep Sequencing in Polycythemia Vera and Essential Thrombocythemia. Blood Adv. 2016; 1:21-30. In some embodiments, a subject having PV has one or more JAK2 mutation in the pseudokinase domain of the JAK2 gene (e.g., exon 12 to exon 15 of the JAK2 gene) (see, e.g., Lee et al., Structural Effects of Clinically Observed Mutations in JAK2 Exons 13-15: Comparison with V617F and Exon 12 Mutations. BMC Struct. Biol. 2009; 9:58). In some embodiments, the JAK2 mutation leads to overactivation of JAK2, which leads to enhanced signaling through STAT5 (Kleppe et. al., JAK-STAT Pathway Activation in Malignant and Nonmalignant Cells Contributes to MPN Pathogenesis and Therapeutic Response. Cancer Discov. 2015; 5:316-331). Under normal conditions, JAK2 mediates signaling by erythropoietin receptor (EpoR) upon binding of EPO to EpoR. Activation of EpoR/JAK2 triggers multiple signaling pathways regulating erythroid precursor survival, proliferation, and differentiation. In some embodiments, the JAK2 mutation results in EPO-independent JAK2/STAT5 activation (James et al., A unique clonal JAK2 mutation leading to constitutive signaling causes polycythaemia vera. Nature. 2005; 434:1144-8; Baxter et al., Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet. 2005; 365:1054-61; Akada et al., Conditional expression of heterozygous or homozygous Jak2V617F from its endogenous promoter induces a polycythemia vera-like disease. Blood. 2010; 115:3589-97). In some embodiments, the EPO-independent JAK2/STAT5 activation leads to continued growth and reproduce of the cell (e.g., hematopoietic progenitor cell) that harbors the mutation. In some embodiments, EPO-independent JAK2/STAT5 activation leads to erythrocytosis. In some embodiments, a subject having PV has a JAK2 exon 14 mutation. In some embodiments, a subject having PV has a JAK2 exon 14 mutation that causes overactivation of JAK2. In some embodiments, a subject having PV has one or more exon 14 mutations including but are not limited to V617F, H606Q, H608Y, L611V, L611S, V617I, C618F, C618R or absent of exon 14 (see, e.g., Regimbeau et al., Genetic Background of Polycythemia Vera, Genes (Basel). 2022 April; 13 (4): 637). In some embodiments, the subject has JAK2 V617F mutation, which is the most prevalent JAK2 mutation in PV patients. The V617F mutation results from a G>T point mutation in JAK2 gene exon 14. The JAK2 V617F mutation results in overactivation of JAK2/STAT5 signaling by destabilization of the inhibitory JH2-JH1 interface and/or contributing to activation of JH1 (Constantinescu et al., Functional Consequences of Mutations in Myeloproliferative Neoplasms, Hemasphere. 2021 Jun. 1; 5 (6): e578). In some embodiments, allele burden of JAK2 V617F mutation affects the severity of PV. In some embodiments, a subject having PV is a heterozygous of the JAK2 V617F mutation. In some embodiments, a subject having PV is a homozygous of the JAK2 V617F mutation. In some embodiments, JAK2 exon 14 mutation (e.g., JAK2 V617F mutation) leads to EPO independent JAK2/STAT5 overactivation. In some embodiments, a PV subject having JAK2 exon 14 mutation (e.g., JAK2 V617F mutation) has lower hepcidin level as compared to subject without PV. In some embodiments, a subject having JAK2 exon 14 mutation can have one or more other mutations described herein.
In some embodiments, a subject having PV has one or more JAK2 exon 12 mutations. In some embodiments, a subject having PV does not have JAK2 V617F mutation. In some embodiments, a JAK2 V617F mutation negative PV subject has one or more mutations in JAK2 exon 12. In some embodiments, JAK2 exon 12 mutations result in overactivation of JAK2/STAT5 pathway. In some embodiments, JAK2 exon 12 mutation results in erythrocytosis. In some embodiments, a subject having PV with JAK2 exon 12 mutation have similar serum and liver iron level compared to a PV subject harboring the JAK2 V617F mutation. In some embodiments, a subject having PV with JAK2 exon 12 mutation have higher hemoglobin levels, higher ERFE level, and even lower hepcidin level compared to PV subjects harboring the JAK2 V617F mutation (see, e.g., Grisouard et al., JAK2 exon 12 mutant mice display isolated erythrocytosis and changes in iron metabolism favoring increased erythropoiesis. Blood. 2016; 128:839-51). In some embodiments, PV subjects having JAK2 exon 12 mutation have similar prognosis as PV subjects with JAK2 V617F mutation (Passamonti et al., Molecular and Clinical Features of the Myeloproliferative Neoplasm Associated with JAK2 Exon 12 Mutations. Blood. 2011; 117:2813-2816). In some embodiments, a subject having PV has one or more exon 12 mutations including but are not limited to F537-K539delinsL, N542-E543del mutation, H538QK539L, V536-1546 dup11, V536-F547 dup, F537-1546dup10F547L, F537IK539I, H538-K539delinsL, H538-K539del, H538DK539LI540S, H538G, K539L, K539E, I540-E543delinsMK, 1540-E542delinsS, R541-E543delinsK, N542-E543del, D544-L545del, or 547insLI540-F547dup8. (see, e.g., Regimbeau et al., Genetic Background of Polycythemia Vera, Genes (Basel). 2022 April; 13 (4): 637).
In some embodiments, a subject having PV has one or more JAK2 exon 13 mutations. In some embodiments, JAK2 exon 13 mutation results in overactivation of JAK2/STAT5 pathway. In some embodiments, JAK2 exon 13 mutations result in erythrocytosis. In some embodiments, a PV subject having JAK2 exon 13 mutation has lower hepcidin level as compared to subject without PV. In some embodiments, a subject having PV has one or more exon 12 mutations including but are not limited to R564L, R564Q, V567A, G571S, G571R, L579F, H587N, S591L, or F557L (see, e.g., Regimbeau et al., Genetic Background of Polycythemia Vera, Genes (Basel). 2022 April; 13 (4): 637).
In some embodiments, a subject having PV has a JAK2 exon 15 mutation. In some embodiments, JAK2 exon 15 mutation results in overactivation of JAK2/STAT5 pathway. In some embodiments, JAK2 exon 15 mutations result in erythrocytosis. In some embodiments, a PV subject having JAK2 exon 15 mutation has lower hepcidin level as compared to subject without PV. In some embodiments, a subject having PV has a 1645V or L642P mutation in JAK2 exon 15 (see, e.g., Regimbeau et al., Genetic Background of Polycythemia Vera, Genes (Basel). 2022 April; 13 (4): 637; Ma et al., Mutation Profile of JAK2 Transcripts in Patients with Chronic Myeloproliferative Neoplasias. J. Mol. Diagn. 2009; 11:49-53).
In some embodiments, a subject having PV has a non-JAK2 mutation that results in JAK2/STAT5 overactivation. For example, a subject having PV may have mutation in the Lymphocyte adaptor protein (LNK) gene (also known as SH2B Adaptor Protein 3 (SH2B3 gene). LNK is a negative regulator of JAK2 in cells (e.g., hematopoietic progenitor cells). In some embodiments, LNK can also modulate signaling mediated by lineage-specific cytokines (e.g., TPO and EPO), thus controlling megakaryocytic and erythroid development, respectively. In some embodiments, LNK negatively modulates EPO receptor (EpoR) signaling by inhibiting pathways in primary erythroblasts (e.g., JAK2/STAT5 pathway). In some embodiments, a PV subject has mutations in the LNK gene that leads to loss of function of LNK, which then leads to overactivation of JAK2/STAT5 pathway (see, e.g., Tefferi et al., Targeted Deep Sequencing in Polycythemia Vera and Essential Thrombocythemia. Blood Adv. 2016; 1:21-30; McMullin et al., LNK Mutations and Myeloproliferative Disorders. Am. J. Hematol. 2016; 91:248-251). In some embodiments, a PV subject having LNK mutation does not have JAK2 mutation. In some embodiments, a PV subject having LKN mutation has concurrent JAK2 mutation (e.g., JAK2 V617F mutation).
In some embodiments, a subject having PV has one or more non-JAK2 mutations including but are not limited to SRSF2 gene, SF3B1 gene, U2AF1 gene, U2AF1 gene, ZRSR2 gene, TET2 gene, DNMT3a gene, IDH1/IDH2 gene, ASXL1 gene, EZH2 gene, LNK/SH2B3 gene, NF-E2 gene, NF1 gene, CBL gene, FLT3 gene, ERBB gene, PPMID gene, TR53 gene, RUNX1 gene, CUX1 gene, ETV6 gene, CALR gene, MPL gene, let-7a gene, miR-26b gene, miR-27b gene, miR-28 gene, miR-30b gene, miR-30c gene, miR-125-5p gene, miR-125b-5p gene, miR-143 gene, miR-145 gene, miR-150 gene, miR-182 gene, miR-223 gene, miR-342 gene, or miR-451 gene. In some embodiments, non-JAK2 mutation can be present in subject having JAK2 mutation (e.g., JAK2 V617F or exon 12 mutation) (see, e.g., Regimbeau et al., Genetic Background of Polycythemia Vera, Genes (Basel). 2022 April; 13 (4): 637).
In some embodiments, a mutation that results in JAK2/STAT5 overactivation (e.g., any one of the mutations described herein) is an acquired mutation. In some embodiments, a mutation that results in JAK2/STAT5 overactivation (e.g., any one of the mutations described herein) is a somatic mutation. In some embodiments, a subject having PV acquired a mutation that causes JAK2/STAT5 overactivation (e.g., any one of the mutations described herein) in a cell in the bone marrow. In some embodiments, a subject having PV acquired a mutation that causes JAK2/STAT5 overactivation (e.g., any one of the mutations described herein) in a hematopoietic progenitor cell. In some embodiments, a subject having PV acquired a mutation that causes JAK2/STAT5 overactivation (e.g., any one of the mutations described herein) in a CD34+CD38− hematopoietic progenitor cell. In some embodiments, a subject having PV acquired a mutation that causes JAK2/STAT5 overactivation (e.g., any one of the mutations described herein) in myeloid progenitor cells. In some embodiments, a subject having PV acquired a mutation that causes JAK2/STAT5 overactivation (e.g., any one of the mutations described herein) in Megakaryocytic-erythroid progenitor cells (see, e.g., Jamieson et al., The JAK2 V617F mutation occurs in hematopoietic stem cells in polycythemia vera and predisposes toward erythroid differentiation, Proc Natl Acad Sci USA. 2006 Apr. 18; 103 (16): 6224-6229; Spivak, The polycythemia vera stem cell, Leukemia Suppl. 2014 December; 3 (Suppl 1): S23-S24).
In some aspects, the present disclosure provides a method for treating polycythemia vera (PV) in a subject, wherein the PV is associated with overactivation of JAK2/STAT5 pathway, the method comprising administering to the subject an effective amount of an anti-TMPRSS6 antibody described herein. In some aspects, the present disclosure provides a method for treating polycythemia vera (PV) in a subject having bone marrow that comprises cells with JAK2/STAT5 overactivation, the method comprising administering the subject an effective amount of anti-TMPRSS6 antibody described herein.
In some embodiments, administration of an anti-TMPRSS6 antibody described herein results in increased circulating hepcidin or pro-hepcidin level (e.g., increasing circulating hepcidin or pro-hepcidin level by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times or at least 100 times) in a subject having PV (e.g., subject having PV associated with JAK2/STAT5 overactivation) relative to the subject prior to receiving the anti-TMPRSS6 antibody or a subject having PV (e.g., subject having PV associated with JAK2/STAT5 overactivation) but not receiving the anti-TMPRSS6 antibody. In some embodiments, administration of an anti-TMPRSS6 antibody described herein results in increased circulating hepcidin or pro-hepcidin level (e.g., increasing circulating hepcidin or pro-hepcidin level by up to 5%, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95%, up to 100%, up to 1.5 times, up to 2 times, up to 3 times, up to 4 times, up to 5 times, up to 10 times, up to 20 times, up to 50 times or up to 100 times) in a subject having PV (e.g., subject having PV associated with JAK2/STAT5 overactivation) relative to the subject prior to receiving the anti-TMPRSS6 antibody or a subject having PV (e.g., subject having PV associated with JAK2/STAT5 overactivation) but not receiving the anti-TMPRSS6 antibody. In some embodiments, increasing hepcidin in subjects having PV (e.g., subject having PV associated with JAK2/STAT5 overactivation) results in augmentation of iron restricted erythropoiesis.
In some embodiments, administration of an anti-TMPRSS6 antibody results in decreased liver iron (e.g., decreasing liver iron by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) in a subject having PV (e.g., subject having PV associated with JAK2/STAT5 overactivation) relative to the subject prior to receiving the anti-TMPRSS6 antibody or a subject having PV (e.g., subject having PV associated with JAK2/STAT5 overactivation) but not receiving the anti-TMPRSS6 antibody. Liver iron level can be evaluated by appropriate lab tests, e.g., Magnetic resonance imaging (MRI) (e.g., Henninger et al., Practical guide to quantification of hepatic iron with MRI, Eur Radiol. 2020; 30 (1): 383-393), liver biopsy, etc.
In some embodiments, administration of an anti-TMPRSS6 antibody results in decreased circulating iron such as serum iron (e.g., decreasing circulating iron by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) in a subject having PV (e.g., subject having PV associated with JAK2/STAT5 overactivation) relative to the subject prior to receiving the anti-TMPRSS6 antibody or a subject having PV (e.g., subject having PV associated with JAK2/STAT5 overactivation) but not receiving the anti-TMPRSS6 antibody. Circulating iron level can be evaluated by appropriate lab tests, e.g., measurement of iron in serum, transferrin saturation (TSAT), or total iron binding capacity (TIBC).
In some embodiments, administration of an anti-TMPRSS6 antibody results in decreased hematocrit (HCT) (e.g., decreasing HCT by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) in a subject having PV (e.g., subject having PV associated with JAK2/STAT5 overactivation) relative to the subject prior to receiving the anti-TMPRSS6 antibody or a subject having PV (e.g., subject having PV associated with JAK2/STAT5 overactivation) but not receiving the anti-TMPRSS6 antibody. Circulating iron level can be evaluated by appropriate lab tests, e.g., Magnetic resonance imaging (MRI) (e.g., Henninger et al., Practical guide to quantification of hepatic iron with MRI, Eur Radiol. 2020; 30 (1): 383-393), liver biopsy, etc.
In some embodiments, administration of an anti-TMPRSS6 antibody results in decreased red blood cell count (e.g., decreasing red blood cell count by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) in a subject having PV (e.g., subject having PV associated with JAK2/STAT5 overactivation) relative to the subject prior to receiving the anti-TMPRSS6 antibody or a subject having PV (e.g., subject having PV associated with JAK2/STAT5 overactivation) but not receiving the anti-TMPRSS6 antibody. Red blood cell count can be evaluated by appropriate lab tests, e.g., blood smear, cell counter, flow cytometry, etc.
In some embodiments, administration of an anti-TMPRSS6 antibody results in decreased red cell distribution width (RDW) (e.g., decreasing RDW by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) in a subject having PV (e.g., subject having PV associated with JAK2/STAT5 overactivation) relative to the subject prior to receiving the anti-TMPRSS6 antibody or a subject having PV (e.g., subject having PV associated with JAK2/STAT5 overactivation) but not receiving the anti-TMPRSS6 antibody. RDW can be evaluated by appropriate lab tests, e.g., blood test.
In some embodiments, administration of an anti-TMPRSS6 antibody results in decreased erythroid progenitor cells (e.g., decreasing erythroid progenitor cells (e.g., in the bone marrow) by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) in a subject having PV (e.g., subject having PV associated with JAK2/STAT5 overactivation) relative to the subject prior to receiving the anti-TMPRSS6 antibody or a subject having PV (e.g., subject having PV associated with JAK2/STAT5 overactivation) but not receiving the anti-TMPRSS6 antibody. Erythroid progenitor cells can be evaluated by appropriate lab tests, e.g., bone marrow biopsy, flow cytometry, etc.
In some embodiments, administration of an anti-TMPRSS6 antibody results in decreased plasma hemoglobin level (e.g., decreasing plasma hemoglobin level by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) in a subject having PV (e.g., subject having PV associated with JAK2/STAT5 overactivation) relative to the subject prior to receiving the anti-TMPRSS6 antibody or a subject having PV (e.g., subject having PV associated with JAK2/STAT5 overactivation) but not receiving the anti-TMPRSS6 antibody. Plasma hemoglobin level can be evaluated by appropriate lab tests, e.g., blood test, etc.
In some embodiments, administration of an anti-TMPRSS6 antibody results in decreased mean corpuscular volume (MCV) (e.g., decreasing MCV level by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) in a subject having PV (e.g., subject having PV associated with JAK2/STAT5 overactivation) relative to the subject prior to receiving the anti-TMPRSS6 antibody or a subject having PV (e.g., subject having PV associated with JAK2/STAT5 overactivation) but not receiving the anti-TMPRSS6 antibody. MCV can be evaluated by appropriate lab tests, e.g., blood test, etc.
In some embodiments, administration of an anti-TMPRSS6 antibody results in decreased leukocytosis (i.e., white cell count) (e.g., decreasing white blood cell count by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) in a subject having PV (e.g., subject having PV associated with JAK2/STAT5 overactivation) relative to the subject prior to receiving the anti-TMPRSS6 antibody or a subject having PV (e.g., subject having PV associated with JAK2/STAT5 overactivation) but not receiving the anti-TMPRSS6 antibody. White blood cell count can be evaluated by appropriate lab tests, e.g., blood smear, cell counter, flow cytometry, etc.
In some embodiments, administration of an anti-TMPRSS6 antibody results in decreased thrombotic events (TEs) (e.g., decreasing TEs by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) in a subject having PV (e.g., subject having PV associated with JAK2/STAT5 overactivation) relative to the subject prior to receiving the anti-TMPRSS6 antibody or a subject having PV (e.g., subject having PV associated with JAK2/STAT5 overactivation) but not receiving the anti-TMPRSS6 antibody. TEs can be determined by appropriate lab tests and clinical visits.
In some embodiments, administration of an anti-TMPRSS6 antibody results in decreased frequency for phlebotomy (e.g., decreasing the frequency for phlebotomy by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) in a subject having PV (e.g., subject having PV associated with JAK2/STAT5 overactivation) relative to the subject prior to receiving the anti-TMPRSS6 antibody or a subject having PV (e.g., subject having PV associated with JAK2/STAT5 overactivation) but not receiving the anti-TMPRSS6 antibody. The need for phlebotomy can be determined by hematological evaluation of the subject, e.g., testing for HCT, hemoglobin, red blood cell count, bone marrow biopsy, etc. In some embodiments, administration of an anti-TMPRSS6 antibody described herein results in 70% to 95% phlebotomy (e.g., between 70% and 95%, between 75% and 90%, between 80% and 85%, between 85% and 90%) independence in subjects receiving the treatment.
In some embodiments, administration of the anti-TMPRSS6 antibody results in decreased frequency for cytoreductive therapy (e.g., decreasing the frequency for cytoreductive therapy by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) in a subject having PV (e.g., subject having PV associated with JAK2/STAT5 overactivation) relative to the subject prior to receiving the anti-TMPRSS6 antibody or a subject having PV (e.g., subject having PV associated with JAK2/STAT5 overactivation) but not receiving the anti-TMPRSS6 antibody. The need for cytoreductive therapy can be determined by hematological evaluation of the subject, e.g., testing for HCT, hemoglobin, red blood cell count, bone marrow biopsy, etc.
In some embodiments, administration of the anti-TMPRSS6 antibody results in improved therapeutic effects (e.g., decreased HCT, hemoglobin, red blood cell count, improved iron deficiency, etc.) in subjects having PV in need thereof but are refractory to or intolerant of phlebotomy and/or cytoreductive therapy.
In some embodiments, an anti-TMPRSS6 antibody described herein can be administered to the subject in need thereof every week, every two weeks, every three weeks, every month, every two months, or every three-months. In some embodiments, an anti-TMPRSS6 is administered via intravenous injection, subcutaneous injection, intraperitoneal injection. In some embodiments, an anti-TMPRSS6 antibody described herein can be administered in a clinic or self-administered by the subject (e.g., via subcutaneous injection).
In some embodiments, administration of an anti-TMPRSS6 antibody results in reduction from baseline in Myeloproliferative Neoplasm Symptom Assessment Form (MPN-SAF). MPN-SAF is a tool for MPN subjects (e.g., PV patients) to track their symptoms and monitor how they feel overtime. The symptoms being monitored include but are not limited to fatigue, filling up quickly when eating (early satiety), abdominal discomfort, inactivity, problems with concentration, night sweats, itching (pruritus), bone pain, fever (higher than 100 F), and unintentional weight loss last six months. In some embodiments, it is expected that more than 45% (e.g., more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, or 100%) of the subjects receiving anti-TMPRSS6 antibody treatment have higher than 50% (e.g., higher than 55%, higher than 60%, higher than 65%, higher than 70%, higher than 75%, higher than 80%, higher than 85%, higher than 90%, higher than 95%, or 100%) reduction from baseline of each subject according to MPN-SAF.
In some embodiments, administration of an anti-TMPRSS6 antibody has or minor adverse effects to the subject receiving the treatment, for example, no hematological adverse events such as thrombocytopenia or neutropenia, no anti-drug antibody, etc.
In some embodiment, the present disclosure provides method of treating a subjecting having PV by administering the subject an effective amount of the anti-TMPRSS6 antibody in combination with any known therapeutics for treating PV, e.g., interferon (e.g., ropeginterferon a-2b-njft (Besremi), pegylated interferon), JAK2 inhibitor (e.g., ruxolitinib, XL019, fedratinib (SAR302503), momelotinib), JAK1 inhibitor (e.g., itacitinib), hepcidin memetic (e.g., rusfertide (PTG-300)), lysine specific demethylase inhibitor (e.g., Bomedemstat (IMG-7298), TMPRSS6 antagonist (e.g., Sapablursen (ISIS 702843), SLN124), anti-TfR1 antibody (e.g., PPMX-T003), MDM2 inhibitor (e.g., Idasanutlin (RG7388), KRT-232), tyrosine kinase inhibitor (e.g., Dasatinib, Erlotinib, Gleevec, lestaurtinib (CEP-701)), HDAC inhibitor (e.g., Givinostat (ITF2357), MK-0683), PI3K inhibitor (e.g., Umbralisib (TGR-1202), telomerase inhibitor (e.g., Imetelstat), phlebotomy, low-dose aspirin, or hydroxyurea.
Antibodies and antibody fragments that can relieve one or more symptoms of a myeloproliferative disorder in vivo when administered to a subject exhibiting an animal model of the disease, i.e., a subject that is known or suspected to have a myeloproliferative disorder, are expected to have therapeutic effectiveness for clinical use. As demonstrated by exemplary embodiments shown in Example 9 using the Jak2V617/+ Vav-iCre mouse model of PV, administration of the anti-TMPRSS6 recombinant monoclonal antibody MWTx-003 resulted in multiple in vivo effects including but not limited to dose-dependent reductions in the hematocrit (HCT) level, reduced circulating red blood cell (RBC) count, and hemoglobin (HGB) concentrations indicating reduced erythrocytosis, as well as increased hepcidin levels, decreased serum iron concentrations, and differential effects on spleen and liver wherein administration of the anti-TMPRSS6 recombinant monoclonal antibody MWTx-003 did not cause major changes in liver iron content, but caused a significant increase in iron deposits in splenic macrophages, compared with isotype controls. Certain effects can be understood as an amelioration of a symptom of the disorder. Symptoms of the disorder are manifested in multiple biological systems that include but are not limited to effects in the liver, in the spleen, in the blood (in particular serum hepcidin levels, RBC, HCT, erythrocytosis), and in the bone marrow. Administration of anti-TMPRSS6 antibodies ameliorated multiple symptoms throughout the disease model subject, shifting the measured symptom levels away from levels seen in isotype controls for the disease model (untreated disease) and towards the levels seen in wildtype littermates that represent normal levels in a genetically similar subject that is not known or suspected to have the disease. The present non-limiting exemplary embodiment discloses an anti-TMPRSS6 antibody therapy that increased hepcidin levels and reduced erythrocytosis in subjects suffering from PV.
Compositions are provided that comprise the anti-TMPRSS6 antibody of the present invention with safe and effective amounts and pharmaceutically acceptable carrier(s) or excipient(s) suitable for the intended use(s) of each composition. Such carriers include but are not limited to: saline, buffer, glucose, water, glycerol, ethanol, excipient, stabilizer, preservative, or combinations thereof. It is understood that the pharmaceutical preparation should match the administration mode.
Anti-TMPRSS6 antibodies disclosed herein can be administered by any suitable means, including but not limited to injection or parenteral infusion. Parenteral infusion can include intramuscular, intravenous, intraarterial, intraperitoneal, subcutaneous administration, or parenteral delivery to the liver. Anti-TMPRSS6 antibodies disclosed herein can be formulated for introduction into hepatic tissue or vasculature for delivery localized to target tissues. Anti-TMPRSS6 antibodies disclosed herein can be administered using a device, or as a depot, or in a sustained-release preparations (e.g., semipermeable matrices of solid hydrophobic polymers containing the antibody, or microcapsules) to allow slow and/or measured and/or localized delivery. Anti-TMPRSS6 antibodies disclosed herein can be formulated and administered using colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
Methods are provided for treating a disorder of iron metabolism using an effective amount of an anti-TMPRSS6 antibody disclosed herein. Without wishing to be bound by a particular mechanism of action, methods provided for targeting TMPRSS6 using anti-TMPRSS6 antibodies disclosed herein result in multiple downstream effects, in particular effects on components (molecules, systems, processes) involved in iron metabolism and erythropoiesis. Without wishing to be bound by a particular mechanism of action, methods are provided for treating a disorder of iron metabolism using an effective amount of an anti-TMPRSS6 antibody disclosed herein to modulate the activity of a component involved in iron metabolism. In particular, methods are provided for treating iron overload disorders associated with excess iron accumulation in tissues and organs, including disorders related to or characterized by ineffective erythropoiesis that may include but are not limited to β-thalassemia, in particular non-transfusion dependent thalassemia, MDS (myelodysplastic syndrome), dyserythropoietic anemia, and sideroblastic anemia. Without being limited to a single mechanism of action, methods are provided for treating iron overload disorders associated with low hepcidin levels, in particular disorders associated with suppressed hepcidin expression, including a disease or state in which abnormal suppression of hepcidin expression is involved, by administering anti-TMPRSS6 antibodies capable of increasing hepcidin expression. Methods are provided for treating myeloproliferative disorders. Methods are provided for treating polycythemia vera (PV). Methods are provided for treating polycythemia vera (PV) associated with insufficient hepcidin suppression.
Methods for treating a disorder of iron metabolism, as provided herein comprise administering an effective amount of an anti-TMPRSS6 antibody disclosed herein to a subject in need thereof, wherein administration of the effective amount of anti-TMPRSS6 antibody ameliorates at least one biological effect (symptom) associated with the disorder. Methods for treating a disorder of iron metabolism associated with suppressed hepcidin levels are provided wherein administration of an effective amount of an anti-TMPRSS6 antibody disclosed herein to a subject in need thereof, results in at least one of increased hepcidin promoter activity, increased hepcidin transcription, increased hepcidin RNA levels, and increased hepcidin levels, in particular serum hepcidin levels. Methods for treating a subject known or suspected to have an iron overload disorder are provided wherein administration of an effective amount of anti-TMPRSS6 antibody results in one or more biological effects including but not limited to reducing liver non-heme iron, increasing serum hepcidin, increasing liver hepcidin RNA, reducing splenomegaly, increasing red blood count (RBC), increasing hematocrit (HCT), reducing red cell distribution width (RDW), and increased production of mature red cells (increased erythropoiesis). Methods for treating a subject known or suspected to have an iron overload disorder characterized by ineffective erythropoiesis are provided wherein administration of an effective amount of anti-TMPRSS6 antibody results in one or more biological effects including but not limited to reducing liver non-heme iron, increasing serum hepcidin, increasing liver hepcidin RNA, reducing splenomegaly, increasing red blood count (RBC), increasing hematocrit (HCT), reducing red cell distribution width (RDW), and increased production of mature red cells (increased erythropoiesis).
Methods and compositions are provided for treating a disorder of iron metabolism, in particular an iron overload disorder, even more particularly an iron overload disorder characterized by ineffective erythropoiesis, wherein administration of an effective amount of an anti-TMPRSS6 antibody results in treating or ameliorating more than one biological effect or symptom associated with the disorder. Without wishing to be bound by a theory or mechanism of action, it is understood that ineffective erythropoiesis characterized by erythroid precursor apoptosis resulting in few mature red cells produced in the bone marrow, is a driving force for abnormal hepcidin suppression leading to increased iron absorption and iron overload. In accordance with this understanding, a treatment that improves erythroblast differentiation and maturation into red cells should be therapeutically beneficial for treating an iron overload disorder. The effectiveness of anti-TMPRSS6 antibody therapy to increase erythroblast differentiation and maturation into red cells, decrease iron loading, increase hepcidin expression, etc., maximizes the therapeutic benefit of the methods and compositions using anti-TMPRSS6 antibodies disclosed herein.
Methods and compositions are provided for treating a myeloproliferative disorder, in particular a myeloproliferative neoplasm such as a chronic myeloproliferative neoplasm, more particularly a myeloproliferative neoplasm characterized by erythroid hyperplasia, even more particularly polycythemia vera (PV), wherein administration of an effective amount of an anti-TMPRSS6 antibody results in treating or ameliorating more than one biological effect or symptom associated with the disorder. Without wishing to be bound by a theory or mechanism of action, the observation of insufficiently suppressed hepcidin in PV patients, in view of the degree of iron deficiency observed in PV patients, is understood to suggest that disordered or dysregulated iron metabolism is an important component of the pathobiology of PV, and in particular that insufficiently suppressed hepcidin levels is component of the pathobiology of PV. In accordance with this understanding, a treatment that modulates hepcidin expression should be therapeutically beneficial for treating a myeloproliferative neoplasm characterized by erythroid hyperplasia, in particular polycythemia vera (PV). The effectiveness of anti-TMPRSS6 antibody therapy to reduce erythrocytosis and normalize hematocrit (HCT) level, and increase hepcidin expression, inter alia, maximizes the therapeutic benefit of the methods and compositions using anti-TMPRSS6 antibodies disclosed herein.
The following examples are offered to illustrate, but not to limit, the claimed invention.
The production of novel monoclonal antibodies against TMPRSS6 was carried out under contract by the LakePharma Discovery Immunology group (LakePharma, Inc. San Carlos, CA), utilizing in vivo rodent immunization and hybridoma technology. DNA-based immunization via hydrodynamic gene transfer tail vein injection was performed in B6;SJL mice (The Jackson Laboratories) using a mixture of pLEV113_huTMPRSS6 and pLEV113_moTMPRSS6-TCE plasmid DNA (cloned at LakePharma, Inc). Sufficient plasma titers as determined by fluorescence-activated cell sorting (FACS) were obtained, triggering downstream antibody recovery and screening activities. Electrofusion using a NEPA GENE ECFG21 Super Electro Cell Fusion Generator (Nepa Gene Co., Ltd., Ichikawa-City, Chiba, Japan) was performed with pooled splenocytes from 2 immunized mice and a myeloma fusion partner. Fusion material was plated in a total of ten (10) 384-well plates in hypoxanthine-aminopterin-thymidine medium, which specifically selects for hybridomas over unfused myeloma partner cells. Hybridoma supernatants were initially screened for HuTMPRSS6 reactivity by FACS measurement to detect supernatants that gave a positive staining signal on TMPRSS6-expressing HEK293T cells (a plasmid encoding huTMPRSS6-(His) 6 (SEQ ID NO: 97) was transfected in HEK293T cells, TMPRSS6-expressing HEK293T cells were selected) and negative staining on parentals (HEK293T) on day 10 post-fusion. Hybridoma supernatants giving a positive staining signal on TMPRSS6-expressing HEK293 cells and negative staining on parentals were designated as “hits” for further screening. 192 hits were identified in the primary FACS screen and 143 hits were confirmed in secondary and tertiary FACS screens.
A hepcidin promoter-luciferase reporter assay was used to measure responses of the HAMP promoter to various anti-TMPRSS6 antibodies (Du, X. et al., 2008. Science 320:1088-1092; modified to use human HAMP promoter instead of mouse Hamp promoter as originally disclosed). For the HAMP-luciferase report assay, a 2.5 kb HAMP promoter fragment (Reference Genome GRCh38) was spliced upstream from a sequence encoding firefly luciferase. A control construct encoding Renilla luciferase, driven by a thymidine kinase promoter (Promega, E6931) was used as an internal control. These constructs were co-transfected into HepG2 cells (ATCC, HB-8065), together with constructs encoding TMPRSS6. Transfected HepG2 cells expressing TMPRSS6 were pre-treated with various concentrations of purified mAb diluted in starvation medium containing minimum essential medium (MEM, ATCC)+1% heat inactivated fetal bovine serum (FBS, Gibco)+1 mM sodium pyruvate+non-essential amino acids solution (Gibco)+10 mM HEPES (Gibco)+1% Pen/Strep (Gibco) for about 3 hrs before treatment with recombinant hBMP6 (R&D Systems) at a final concentration of 25-60 ng/ml to trigger BMP-SMAD-mediated signaling. Purified mouse IgG (Sigma-Aldrich) or human IgG1 (BioXcell) was used as a control. Upon an overnight treatment of hBMP6, cells were lysed and luciferase substrate were added. Luminescence readings from firefly luciferase and Renilla luciferase were each recorded by measuring total luminescence. Activity was calculated as the ratio of firefly luciferase luminescence to Renilla luciferase luminescence (control). Results for these assays are shown in
To screen for functionally active hybridomas, the HAMP-luciferase reporter assay described above was used to test all 143 HuTMPRSS6 binding hybridomas (“hits”). Supernatants of ten (10) out of 143 HuTMPRSS6 binding hybridomas increased HAMP promoter activity (data not shown), and were identified as “active clones” to undergo further testing. These ten (10) active clones were tested for cross reactivity against murine target MoTMPRSS6 as described in Example 4 below, and three (3) showed binding towards both HuTMPRSS6 and MoTMPRSS6 as measured by FACS. These three cross-reactive clones were further plated at a density of 1 cell/well in 192 wells of 384-well plates to generate monoclonal hybridoma clones, the resulting subclones that exhibited desired functional activity and cross-reactivity against non-human targets, e.g. murine TMPRSS6 (moTMPRSS6) and/or cynomolgus monkey TMPRSS6 (cynoTMPRSS6) were identified as MWTx-001, MWTx-002, and MWTx-003.
Sequences of MWTx-001, MWTx-002, and MWTx-003 were determined by isolating mRNAs from each hybridoma sample, carrying out reverse transcription polymerase chain reaction (RT-PCR) with unique mouse IgG-specific primer sets to amplify the target variable regions for sequencing. A unique heavy chain and a unique light chain were identified for each anti-TMPRSS6 antibody. The nucleotide sequence of each heavy chain and each light chain was determined. Amino acid sequences encoded by the nucleotide sequences were determined, CDR regions were identified using the Kabat numbering system. Table 1 presents heavy chain and light chain variable region amino acid sequences, and amino acid sequences of identified CDRs (based on Kabat numbering) and heavy chain and light chain variable region nucleotide sequences for each of MWTx-001, MWTx-002, and MWTx-003.
Humanization of the parental antibody was performed utilizing CDR grafting onto human antibody frameworks. Homology modeling of the parental antibody's 3-dimensional structure was first performed to establish a structural model of the parental antibody. Amino acid sequences for the variable fragment framework were identified based on the overall sequence identity, matching VH-VL interface positions, similarly classed CDR canonical positions, and removal of potential N-glycosylation sites. Humanized antibodies were designed by creating multiple hybrid sequences that fuse selected parts of the parental antibody sequence with the human framework sequences. The isotypes chosen to format humanized antibody were IgG1 for the heavy chain and IgG1 kappa for the light chain. Using the 3D model, these humanized sequences were methodically analyzed by eye and computer modeling to isolate the sequences that would most likely retain antigen binding. The goal was to maximize the amount of human sequence in the final humanized antibodies while retaining the original antibody specificity. Humanized variants, pairing the humanized VH and VL were then expressed and purified for affinity analysis.
In one round of designing, generating, and testing variants as part of an affinity analysis, four VH variants were generated with the VH-CDRs of the parental antibody MWTX-003 in corresponding positions in four different human IgG1-derived frameworks (SEQ ID NOS: 89-92), and four VL (VK) variants were generated with the VL-CDRs of the parental antibody MWTX-003 in corresponding positions in four different human IgG1 kappa-derived frameworks (SEQ ID NOS: 93-96). A total of sixteen (16) humanized variants representing every combination of the VH and VL (VK) variants were prepared according to a 4VH×4VK matrix, evaluated for antigen binding characteristics (kon, koff, KD) and found to have KD values in the nanomolar range, from 4.16E-07 (to 1.09E-08.
Variants that showed desired antigen binding affinity were selected for further evaluation and development. In some cases, parental CDR sequences were modified to avoid potential unwanted events such as aspartate isomerization.
To silence antibody effector function, in particular to silence antibody-dependent cellular cytotoxicity (ADCC), critical amino acid residues in the Fc region were identified and mutated (substituted) for all of the humanized antibody variants. Guidance available in the published literature concerning Fc mutations to achieve the goal of abolishing ADCC was used to inform the present mutations, for example removal of the native Fc N-linked glycosylation site (N297A mutation) in hlgG1, or substitutions of leucine at positions 234 and 235 of the lower hinge region in the Fc (LALA double mutation) as described by (Tamm A, Schmidt R E. IgG binding sites on human Fc gamma receptors. Int Rev Immunol. 1997; 16 (1-2): 57-85. doi: 10.3109/08830189709045703; Jefferis R, Lund J. Interaction sites on human IgG-Fc for FcgammaR: current models. Immunol Lett. 2002 Jun. 3; 82 (1-2): 57-65. doi: 10.1016/s0165-2478 (02) 00019-6). In the present variants, the N297A mutation was introduced in the Fc of the hzMWTx-001 Var and hzMWTx-002 Var antibodies, and the LALA mutation was introduced into the Fc of the hzMWTx-003 Var antibody, to achieve the same goal of reducing or silencing ADCC (Table 3, SEQ ID NOs: 73, 77, 81).
After evaluation, humanized anti-TMPRSS6 antibody variants hzMWTx-001 Var, hzMWTx-002 Var, and hzMWTx-003 Var were selected for further testing. Sequences and features of humanized variants are shown in Tables 2 and 3 below.
Expression constructs for humanized anti-TMPRSS6 antibody variants were engineered with internal ribosome entry site (IRES) between LC- and HC-coding DNA sequences, codon optimized by Geneart DNA synthesis and cloned into pcDNA3.4 mammalian expression vector (ThermoFisher). The sequences of DNA inserts were verified by sequencing. For recombinant antibody production, the expression constructs were used for transient transfection using ExpiCHO expression system (ThermoFisher) following manufacturer's instruction. The expressed antibodies were purified by Protein A affinity chromatography. The yield of antibody production from transient transfection ranged from 50 mg to 300 mg per liter, with purity>95% and <1 EU/ml endotoxin level.
Sequences of Humanized Anti-TMPRSS6 Antibody Variants hzMWTx-001 Var, hzMWTx-002 Var, and hzMWTx-003 Var
Humanized anti-TMPRSS6 antibody variants hzMWTx-001 Var, hzMWTx-002 Var, and hzMWTx-003 Var were selected for further testing. Sequences of the variable region of each variable region are shown in Table 2 below, where identified CDRs are indicated by underlining and changes made in the humanized variant CDR sequences relative to the parental antibody are indicated and discussed.
Table 3 shows complete heavy chain and light chain protein and nucleotide sequences of anti-TMPRSS6 monoclonal antibodies MWTx-001, MWTx-002, and MWTx-003, and humanized anti-TMPRSS6 antibody variants hzMWTx-001 Var, hzMWTx-002 Var, and hzMWTx-003 Var. Heavy chain protein sequences of humanized anti-TMPRSS6 antibody variants hzMWTx-001 Var, hzMWTx-002 Var, and hzMWTx-003 Var show the location of mutations (changes) introduced to reduce ADCC as described above.
AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSS
SVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDV
LMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMS
GKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYV
EWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSR
TPG (SEQ ID NO: 61)
TSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERH
NSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 63)
SVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIF
PPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQ
HQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNP
GDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLK
KTISRSPGK (SEQ ID NO: 65)
QLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYE
RHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 67)
PSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVP
SSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIK
DVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQD
WMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDIS
VEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISR
SPGK (SEQ ID NO: 69)
TSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERH
NSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 71)
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV
VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT
LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
A
STYRVVSVLTVLHQDWL
NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS
PG (SEQ ID NO: 73)
SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
YACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 75)
LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK
PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
A
STYRVVSVLTVLH
QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ
KSLSLSPG (SEQ ID NO: 77)
QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEK
HKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 79)
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS
VVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE
AA
GGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS
LSLSPG (SEQ ID NO: 81)
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH
KVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 83)
The binding affinity of various anti-TMPRSS6 antibodies to human TMPRSS6 expressed on HEK293T cells was measured using three different methods: cell surface ELISA (
HEK293T cells stably expressing human TMPRSS6 (generated by LakePharma Inc as described above; SEQ ID NO: 97) were fixed with 4% paraformaldehyde (PFA), and washed with dPBS (Dulbecco's phosphate-buffered saline, Corning Cellgro) before incubation with various concentrations of anti-TMPRSS6 antibodies diluted in BSA medium (DMEM+1% Pen/Strep+10 mM HEPES+1 mg/ml BSA (Sigma-Aldrich). Purified mouse IgG was used as a control (Sigma-Aldrich). After incubation, cells were washed with BSA medium and then incubated with goat anti-mouse IgG conjugated with HRP as a 2° antibody (Invitrogen). At last, cells were washed with dPBS to remove unbound antibody and color developed with ELISA liquid substrate (Sigma-Aldrich), followed by stopping the reaction with addition of the same volume of ELISA liquid substrate of 1M H2SO4. Bound antibody was measured by absorbance at OD450nm. Results for these assays are shown in
HEK293T cells stably expressing human TMPRSS6 were collected, and blocked with dPBS+3% BSA before incubation with various concentrations of anti-TMPRSS6 antibodies diluted in dPBS+3% BSA. Purified mouse IgG was used as a control. After incubation, cells were washed with dPBS and then incubated with goat anti-mouse IgG conjugated with APC as a 2° antibody (Jackson ImmunoResearch Inc). At last, cells were washed with dPBS to remove unbound antibody, re-suspended with dPBS+1 mM EDTA, and then subjected to FACS analysis using a NOVOCYTER Flow Cytometer (ACEA Biosciences, Inc., San Diego CA). Bound antibody was determined by measuring mean APC intensity after excitation at 640 nm and measurement of emission (fluorescence) at 675 nm. Results for these assays are shown in
Bio-Layer Interferometry technology was used for anti-TMPRSS6 antibody affinity measurement and binding kinetics determinations with Octet® RED96e system (Sartorius AG). Pre-hydrated Anti-Mouse IgG Fc Capture (AMC) biosensors (for MWTx-001, MWTx-002 and MWTx-003 anti-TMPRSS6 antibodies,
Selected anti-TMPRSS6 antibodies were tested to determine whether any are capable of binding to TMPRSS6 from mouse and/or cynomolgus monkey. HEK293T cells stably expressing human TMPRSS6 (HuTMPRSS6-(His)6) (generated by LakePharma Inc as described above), HEK293T cells stably expressing mouse TMPRSS6 (MoTMPRSS6-(His)6) (SEQ ID NO: 98) (generated by LakePharma Inc as described above) and HEK293T cells transiently expressing cynomolgus monkey TMPRSS6 (CynoTMPRSS6-(His)6) (SEQ ID NO: 99) (generated in house) were collected. HEK293T cells stably expressing human TMPRSS6 were used as a positive control and HEK293T cells were used as a negative control (as described above). Cells were blocked with dPBS+3% BSA before incubation with anti-TMPRSS6 antibodies diluted in dPBS+3% BSA. After incubation, cells were washed with dPBS and followed by another incubation with goat anti-mouse IgG conjugated with AlexaFluor-488 as a 2° antibody (Invitrogen). At last, cells were washed with dPBS to remove unbound antibody, re-suspended with dPBS+1 mM EDTA, and then subjected to FACS analysis using a NOVOCYTE® Flow Cytometer (ACEA Biosciences, Inc., San Diego CA). Bound antibody was determined by excitation at 488 nm and measurement of emission (FITC-A) at 530 nm. Results for these assays are shown in histogram plots in
HEK293T cells stably expressing mouse TMPRSS6 (generated by LakePharma Inc as described above,
To determine if anti-TMPRSS6 antibodies bind to homologous matriptases, HEK293T cells over-expressing matriptase (ST14) (SEQ ID NO: 100) (
In order to study the in vivo pharmacodynamic responses of anti-TMPRSS6 antibodies, 2-10 mg/kg of MWTx-003 anti-TMPRSS6 antibody (
Effects of Treatment with Anti-TMPRSS6 Antibodies on Serum Iron
Serum iron was measured by a chromogenic assay developed in house (
Effects of Treatment with Anti-TMPRSS6 Antibodies on Serum Hepcidin
Serum hepcidin was measured by Hepcidin-Murine Compete ELISA kit purchased from Intrinsic Lifesciences (La Jolla, CA) according to the manufacturer's instructions (
Effects of Treatment with Anti-TMPRSS6 Antibodies on Liver Hepcidin RNA
Liver hepcidin RNA was quantified by real-time qPCR (
Serum concentration of MWTx-003 anti-TMPRSS6 antibody or its humanized variant hzMWTx-003 Var anti-TMPRSS6 antibody was quantified by cell surface ELISA developed in house (as described above,
In order to study in vivo efficacy of anti-TMPRSS6 antibody, a β-thalassemia mouse model (B6.129P2-Hbb-b1tm1Unc Hbb-b2tm1Unc/J, JAX Stock No: 002683, The Jackson Laboratories, Bar Harbor ME) was chosen, herein referred to as Th3/+ mouse. 4-5 weeks old Th3/+ mice and their wildtype (WT) littermates were put on an iron sufficient diet (Teklad TD.80394) and Th3/+ mice were treated with 10 mg/kg MWTx-003 anti-TMPRSS6 antibody or mouse IgG2b isotype control every three days for 4 weeks, while WT littermates did not receive treatments. At the end of the treatment course, mice were euthanized, and spleen, liver, femur and blood samples were collected. Liver total RNA was purified, and serum was collected as described above.
Complete Blood Count (CBC) was performed by VETSCAN HM5 automated hematology analyzer (
Spleen weight was measured, and MWTx-003 anti-TMPRSS6 antibody treatment significantly reduced splenomegaly for Th3/+ mice (
Serum iron was measured as described above. Treatment with MWTx-003 anti-TMPRSS6 antibody significantly reduced serum iron (
Serum hepcidin was measured by Hepcidin-Murine Compete ELISA kit as described above. Treatment with MWTx-003 anti-TMPRSS6 antibody significantly increased serum hepcidin (
Liver hepcidin RNA was quantified by real-time qPCR as described above. Treatment with MWTx-003 anti-TMPRSS6 antibody significantly increased liver hepcidin RNA (
Serum concentration of MWTx-003 anti-TMPRSS6 antibody was quantified by cell surface ELISA developed in-house as described above (
In order to study effects of MWTx-003 anti-TMPRSS6 antibody on erythropoiesis in Th3/+ mice, bone marrow was harvested from femur (see
In Th3/+ mice, treatment with MWTx-003 anti-TMPRSS6 antibody improved ineffective erythropoiesis, with a significant proportion of erythroblasts differentiated and matured into red blood cells.
OCTET® RED96e was used for epitope binning of MWTx-001 (
The effects of anti-TMPRSS6 recombinant monoclonal antibody treatment on reversing erythrocytosis and normalizing hematocrit level in a polycythemia vera (PV) mouse model were evaluated.
B6N.129S6 (SJL)-Jak2tm1.1Ble/AmlyJ mouse (JAX #031658), commonly known as Jak2V617F-Fl/+, is a floxed strain having an inverted V617F mutation carrying exon 14 downstream of the endogenous exon 14 of the Janus kinase 2 (Jak2) gene. The V617F mutation is commonly found in patients with myeloproliferative neoplasm and is present in approximately 95% of patients with PV. When bred to mice that express tissue-specific Cre recombinase, resulting offspring will have the floxed endogenous exon 14 removed and the V617F mutant exon 14 placed into correct transcriptional orientation.
B6.Cg-Commd10Tg(Vav1-icre)A2Kio/J mouse (JAX #008610), commonly known as Vav-iCre, expresses an optimized variant of Cre recombinase (iCre) specifically in hematopoietic cells, and is useful for generating conditional mutations in hematopoietic progenitor compartment. The progeny of Jak2V617F-Fl/+ mice crossed with Vav-iCre transgenics can develop PV, characterized by erythrocytosis and elevated hematocrit levels, and the phenotypes can be propagated by transplanting the bone marrow cells from the double transgenic mice into lethally irradiated wild-type recipient mice.
Recombinant mouse anti-TMPRSS6 monoclonal antibody MWTx-003 is designated as r4K12B in this study, where the antibody is a recombinantly expressed version of mouse monoclonal MWTx-003, and can be referred to as recombinant monoclonal antibody MWTx-003, recombinant MWTx-003, or MWT-003 as in
The following materials were used to evaluate the effects of anti-TMPRSS6 antibody treatment on reversing erythrocytosis and normalizing hematocrit level in a polycythemia vera mouse model.
r4K12B, recombinant mouse monoclonal antibody (recombinant MWTx-003), made in house
InVivoPlus mouse IgG2b Isotype Control, purchased from BioXCell (#BP0086)
10-12-week-old wild-type C57BL/6J (JAX #000664) male mice were purchased from The Jackson Laboratory and allowed to acclimate to the housing environment prior to the initiation of the study. All mice received whole body irradiation at a lethal dose of 1000 cGy at 3.45 Gy/min. 24 hours later, 5×106 bone marrow cells isolated from Jak2V617/+ Vav-iCre double transgenic mice (both male and female mice were used) were injected into each lethally irradiated recipient C57BL/6J mouse through lateral tail vein. Antibiotics (sulfamethoxazole and trimethoprim) in acidified drinking water (pH 2.5-3.0) were administered ad libitum immediately after bone marrow transplantation (BMT) for two weeks. BMT animals were monitored for the development of PV phenotypes by Complete Blood Count using an automatic hematology analyzer. Four weeks post BMT, when the PV phenotype was fully established, mice received intraperitoneal injections of anti-TMPRSS6 antibody r4K12B (recombinant MWTx-003) or mouse IgG2b isotype control antibody once every 4 days for a total of 3 weeks. Animals were euthanized 4 days after the final dose, and bone marrow, spleen, liver, and whole blood were harvested for analyses. Effects of the anti-TMPRSS6 antibody treatment on erythroid profiles, hematological parameters (including mean corpuscular volume (MCV) and average RBC size), splenomegaly, and tissue iron deposition of the mice were evaluated.
Serum hepcidin was measured by Hepcidin-Murine Compete™ ELISA (Intrinsic Lifesciences, SKU #HMC-001) according to the manufacturer's instructions as described above. Results are shown in (
Serum iron was measured using a chromogenic assay as described above.
Iron deposition in the spleen and liver was evaluated by Perls' Prussian blue staining on 10% formalin fixed liver and spleen sections. The sectioning, staining, and imaging work were contracted to Reveal Biosciences (San Diego, California). (
Red blood cell indices were analyzed by complete blood count (CBC) on HM5 VetScan Hematology Analyzer. (
Differentiation of erythroblasts was evaluated in bone marrow and spleen, respectively. Bone marrow harvested from femur and splenocytes from spleen were analyzed by FACS as described above. Results are shown in (
Serum concentration of r4K12B anti-TMPRSS6 antibody (recombinant MWTx-003) was quantified by cell surface ELISA developed in house as described above. Results are shown in (
One-way ANOVA was used to compare three or more sets of data using GraphPad Prism software. P<0.05 was considered statistically significant.
Body weights of the bone marrow recipient C57BL/6J mice (all males) were measured during acclimation for randomization to obtain similar average body weight between groups. Group assignment were performed per the following table (Table 4).
Blood samples were collected from recipient mice (wild-type C57BL/6J mice receiving bone marrow transplant (BMT) of Jak2V617/+ Vav-iCre bone marrow cells) and analyzed for hematological parameters at 3-week and 4-week post BMT, respectively. Referenced to Jak2V617/+ Vav-iCre double transgenic mice (designated as “PV reference strain”), the PV phenotype was developed in the recipient mice at 3 weeks post-BMT, and fully established by 4 weeks post BMT (Table 5).
4 weeks post BMT, when the PV phenotype was fully established, mice (designated at “PV phenotype” mice) received intraperitoneal injections of anti-TMPRSS6 antibody r4K12B (recombinant MWTx-003) at 2 mg/kg, 5 mg/kg, and 10 mg/kg dose levels, respectively, or 10 mg/kg mouse IgG2b isotype control once every 4 days for 3 weeks. The end-point analysis was performed 4 days after the final injection.
After 2 weeks' treatment with anti-TMPRSS6 antibody r4K12B, a trend of dose-dependent reduction in the hematocrit (HCT) level, red blood cell (RBC) count, and hemoglobin (HGB) concentration was observed in mice receiving r4K12B, compared with animals treated with isotype control antibody (Table 6).
Results after 3 Weeks of Treatment
At the end of the 3-week treatment period, HCT levels in all r4k12B treated groups were further decreased in a dose-dependent manner to a similar or lower levels than seen in wild-type (WT) untreated animals (
Subchronic treatment with anti-TMPRSS6 antibody substantially reduced erythrocytosis and normalized hematocrit level in the mouse model of polycythemia vera by limiting iron availability to erythroid precursors. Anti-TMRSS6 antibody treatment offers a promising therapeutic approach in the management of PV, where erythrocytosis and high HCT levels are associated with poor outcomes.
This patent application claims priority to U.S. Provisional Patent Application No. 63/504,357, filed on May 25, 2023, the entire contents of which are fully incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63504357 | May 2023 | US |
