Treatment of pathologies associated with beta-thalassemia

Abstract

Provided is a method of treating or alleviating one or more pathologies associated with β- thalassemia in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an effective amount of an anti-matriptase-2 antibody or antigen-binding fragment thereof. The one or more pathologies may be insoluble liver iron stores: α-globin precipitates in ery throid cells; reactive oxidized species formation; apoptosis of erythroid cells; reticulocytosis; or a combination thereof.

Description

TREATMENT OF PATHOLOGIES ASSOCIATED WITH BETA-THALASSEMIA CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 672,038, filed on July 16, 2024, which is herein incorporated by reference in its entirety.SEQUENCE LISTING

[0002] This application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on January 17, 2025, is named IPE_002_WOl-Sequence_Listing and is 49,585 bytes in size.BACKGROUND

[0003] Beta-thalassemia ((J-thalassemia) is a hereditary disorder caused by mutations in the (3-globin gene, which lead to defective or complete lack of production of (3-globin chains of hemoglobin. The resulting excess of free a-globin causes precipitates that adhere to the cell membrane, leading to oxidative stress, red blood cell membrane damage, premature death of late-stage ery throid precursors, and results in ineffective erythropoiesis, z.e., overproduction of erythroid progenitors that fail to generate mature erythrocytes. Patients display considerable clinical heterogeneity. Those with (3- thalassemia major produce no or very little P-globin, require chronic red blood cell transfusions to survive, and therefore develop extreme iron overload secondary' to the transfusions. However, even when transfusions are not required as in patients with (3- thalassemia intermedia, the manifestations of the disease can be severe, including chronic anemia and splenomegaly due to extramedullary' hematopoiesis and increased turnover of damaged red blood cells. Anemia and ineffective erythropoiesis also cause increased intestinal iron absorption mediated by duodenal hypoxia and erythroid-mediated suppression of the iron regulatory hormone hepcidin. The resulting iron overload adds to morbidity’ by causing cirrhosis and cardiomyopathy, and by exacerbating erythroid cell damage, apoptosis, and ineffective erythropoiesis (Ramos et al., 2010; Melchiori et al., 2010).

[0004] The only currently approved therapy for patients with non-transfusion- dependent P-thalassemia (NTDT), iron chelation, is suboptimal and does not specifically address the underlying pathological mechanisms (Musallam et al., 2021). In the last decade, new agents have been designed to ameliorate ineffective erythropoiesis (activin receptor II ligand traps (Suragani et al., 2014a; Suragni et al., 2014b) or to prevent iron overload (Tmprss6 antisense oligonucleotides or siRNAS (Guo et al.. 2013; Schmidt et al.. 2013), minihepcidins (Casu etal., 2016a). ferroportin inhibitors (Manolova et al., 2019). However, these new agents address only single pathophysiological aspects of the disease and do not entirely correct the inefficiency of the erythropoiesis and its follow-on effect. Moreover, antisense oligonucleotides that downregulate Tmprss6 have been found to produce severe adverse effects, such as thrombocytopenia, during the course of several human clinical trials, raising some safety concerns on their long-term use15.

[0005] Well-tolerated and active novel drugs are needed to address the existing challenges in treating or ameliorating p-thalassemia.SUMMARY OF THE INVENTION

[0006] Some of the main aspects of the present invention are summarized below. Additional aspects are described in the Detailed Description of the Invention, Examples, Drawings, and Claims sections of this disclosure. The description in each section of this disclosure is intended to be read in conjunction with the other sections. Furthermore, the various embodiments described in each section of this disclosure can be combined in various different ways, and all such combinations are intended to fall within the scope of the present invention.

[0007] The present invention is based, in part, on a series of important discoveries that are described in more detail in the Examples section of this patent specification. For example, when administered weekly to a mouse model exhibiting a P-thalassemia intermedia phenoty pe (Hbbth3 / + mice), anti-matriptase-2 antibodies induced hepcidin expression, reduced iron loading, prevented the formation of toxic a-chain / heme aggregates, reduced reactive oxygen species (ROS) formation, and improved reticulocytosis and splenomegaly. Further, administration of these antibodies with a ligand-trapping protein that contains the extracellular domain of activin receptor ty pe IIB entirely reversed the P-thalassemia phenotype in Hbb^^ mice and simultaneouslycorrected iron overload, ineffective erythropoiesis, splenomegaly, and hematological parameters.

[0008] Accordingly, in one aspect, the present invention relates to a method of treating one or more pathologies associated with |3-thalassemia in a subject in need thereof, in which the method comprises administering to the subject a pharmaceutical composition comprising an effective amount of an anti-matriptase-2 antibody or antigenbinding fragment thereof. In another aspect, the present invention relates to a pharmaceutical composition comprising an effective amount of an anti-matriptase-2 antibody or antigen-binding fragment thereof for use in treating one or more pathologies associated with |3-thalassemia in a subject in need thereof.

[0009] In another aspect, the present invention relates to a method of alleviating one or more pathologies associated with |3-thalassemia in a subject in need thereof, in which the method comprises administering to the subject a pharmaceutical composition comprising an effective amount of an anti-matriptase-2 antibody or antigen-binding fragment thereof. In yet another aspect, the present invention relates to a pharmaceutical composition comprising an effective amount of an anti-matriptase-2 antibody or antigenbinding fragment thereof for use in alleviating one or more pathologies associated with |3- thalassemia in a subject in need thereof.

[0010] In some embodiments, the one or more pathologies is selected from insoluble liver iron stores; a-globin precipitates in erythroid cells; reactive oxidized species (ROS) formation; apoptosis of erythroid cells; reticulocytosis; and a combination thereof. In certain embodiments, the one or more pathologies is a-globin precipitates in erythrocytes.

[0011] In some embodiments, the one or more pathologies is ROS formation in spleen, bone marrow, or a combination thereof.

[0012] In some embodiments, the one or more pathologies is apoptosis of erythroid cells in spleen, bone marrow, or a combination thereof.

[0013] In embodiments of the invention, the anti-matriptase-2 antibody or antigenbinding fragment thereof comprises (a) a heavy chain variable region comprising: a complementarity determining region (CDR)-1 domain comprising an amino acid sequence of SEQ ID NO. 1, a CDR-2 domain comprising an amino acid sequence of SEQ ID NO. 2, and a CDR-3 domain comprising an amino acid sequence of SEQ ID NO. 3; and (b) a light chain variable region comprising: a CDR-1 domain comprising an aminoacid sequence of SEQ ID NO. 4, a CDR-2 domain comprising an amino acid sequence of SEQ ID NO. 5. and a CDR-3 domain comprising an amino acid sequence of SEQ ID NO. 6.

[0014] In certain embodiments, the anti-matriptase-2 antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region comprising a CDR-1 comprising an amino acid sequence of SEQ ID NO. 7, a CDR-2 domain comprising an amino acid sequence of SEQ ID NO. 8, and a CDR-3 domain comprising an amino acid sequence of SEQ ID NO. 9; and (b) a light chain variable region comprising a CDR-1 domain comprising an amino acid sequence of SEQ ID NO. 10, a CDR-2 domain comprising an amino acid sequence of SEQ ID NO. 11, and a CDR-3 domain comprising an amino acid sequence of SEQ ID NO. 6.

[0015] In some embodiments, the heavy chain variable region comprises an amino acid sequence at least 95% identical to SEQ ID NO. 12, and the light chain variable region comprises an amino acid sequence at least 95% identical to SEQ ID NO. 13. In certain embodiments, the heavy chain variable region comprises an amino acid sequence of SEQ ID NO. 12, and the light chain variable region comprises an amino acid sequence of SEQ ID NO. 13.

[0016] In some embodiments, the anti-matriptase-2 antibody or antigen-binding fragment thereof comprises: heavy chain CDR-1, CDR-2, and CDR-3 domains contained within a heavy chain variable region comprising an amino acid sequence of SEQ ID NO. 12; and light chain CDR-1 , CDR-2, and CDR-3 domains contained within a light chain variable region comprising an amino acid sequence of SEQ ID NO. 13.

[0017] In some embodiments, the heavy chain comprises an amino acid sequence of SEQ ID NO. 14, and the light chain comprises an amino acid sequence of SEQ ID NO. 15.

[0018] In some embodiments, the anti-matriptase-2 antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region comprising: a CDR-1 domain comprising an amino acid sequence of SEQ ID NO. 1, a CDR-2 domain comprising an ammo acid sequence of SEQ ID NO. 2, and a CDR-3 domain comprising an amino acid sequence of SEQ ID NO. 16; and (b) a light chain variable region comprising: a CDR-1 domain comprising an amino acid sequence of SEQ ID NO. 17, aCDR-2 domain comprising an amino acid sequence of SEQ ID NO. 5, and a CDR-3 domain comprising an amino acid sequence of SEQ ID NO. 18.

[0019] In some embodiments, the anti-matriptase-2 antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region comprising a CDR-1 comprising an amino acid sequence of SEQ ID NO. 19, a CDR-2 domain comprising an amino acid sequence of SEQ ID NO. 20, and a CDR-3 domain comprising an amino acid sequence of SEQ ID NO. 9; and (b) a light chain variable region comprising a CDR-1 domain comprising an amino acid sequence of SEQ ID NO. 21, a CDR-2 domain comprising an amino acid sequence of SEQ ID NO. 11, and a CDR-3 domain comprising an amino acid sequence of SEQ ID NO. 18.

[0020] In some embodiments, the heavy chain variable region comprises an amino acid sequence at least 95% identical to SEQ ID NO. 22, and the light chain variable region comprises an amino acid sequence at least 95% identical to SEQ ID NO. 23. In certain embodiments, the heavy chain variable region comprises an amino acid sequence of SEQ ID NO. 22, and the light chain variable region comprises an amino acid sequence of SEQ ID NO. 23.

[0021] In some embodiments, the anti-matriptase-2 antibody or antigen-binding fragment thereof comprises: (a) heavy chain CDR-1, CDR-2, and CDR-3 domains contained within a heavy chain variable region comprising an amino acid sequence of SEQ ID NO. 22; and (b) light chain CDR-1. CDR-2, and CDR-3 domains contained within a light chain variable region comprising an amino acid sequence of SEQ ID NO. 23.

[0022] In certain embodiments, the heavy chain comprises an amino acid sequence of SEQ ID NO. 24, and the light chain comprises an amino acid sequence of SEQ ID NO. 25.

[0023] In some embodiments, the anti-matriptase-2 antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region comprising: a CDR-1 domain comprising an amino acid sequence of SEQ ID NO. 26. a CDR-2 domain comprising an ammo acid sequence of SEQ ID NO. 27. and a CDR-3 domain comprising an amino acid sequence of SEQ ID NO. 28; and (b) a light chain variable region comprising: a CDR-1 domain comprising an amino acid sequence of SEQ ID NO. 29, aCDR-2 domain comprising an amino acid sequence of SEQ ID NO. 5, and a CDR-3 domain comprising an amino acid sequence of SEQ ID NO. 30.

[0024] In some embodiments, the anti-matriptase-2 antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region comprising a CDR-1 comprising an amino acid sequence of SEQ ID NO. 31, a CDR-2 domain comprising an amino acid sequence of SEQ ID NO. 32, and a CDR-3 domain comprising an amino acid sequence of SEQ ID NO. 33; and (b) a light chain variable region comprising a CDR-1 domain comprising an amino acid sequence of SEQ ID NO. 34, a CDR-2 domain comprising an amino acid sequence of SEQ ID NO. 35, and a CDR-3 domain comprising an amino acid sequence of SEQ ID NO. 30.

[0025] In some embodiments, the heavy chain variable region comprises an amino acid sequence at least 95% identical to SEQ ID NO. 36, and the light chain variable region comprises an amino acid sequence at least 95% identical to SEQ ID NO. 37. In certain embodiments, the heavy chain variable region comprises an amino acid sequence of SEQ ID NO. 36; and (b) the light chain variable region comprises an amino acid sequence of SEQ ID NO. 37.

[0026] In some embodiments, the anti-matriptase-2 antibody or antigen-binding fragment thereof comprises: (a) heavy chain CDR-1, CDR-2, and CDR-3 domains contained within a heavy chain variable region comprising an amino acid sequence of SEQ ID NO. 36; and light chain CDR-1, CDR-2, and CDR-3 domains contained within a light chain variable region comprising an amino acid sequence of SEQ ID NO. 37.

[0027] In certain embodiments, the heavy chain comprises an amino acid sequence of SEQ ID NO. 38, and the light chain comprises an amino acid sequence of SEQ ID NO. 39.

[0028] In some embodiments, the anti-matriptase-2 antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region comprising: a CDR-1 domain comprising an amino acid sequence of SEQ ID NO. 40, a CDR-2 domain comprising an amino acid sequence of SEQ ID NO. 41, and a CDR-3 domain comprising an ammo acid sequence of SEQ ID NO. 42; and (b) a light chain variable region comprising: a CDR-1 domain comprising an amino acid sequence of SEQ ID NO. 43, a CDR-2 domain comprising an amino acid sequence of SEQ ID NO. 44, and a CDR-3 domain comprising an amino acid sequence of SEQ ID NO. 45.

[0029] In some embodiments, the anti-matriptase-2 antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region comprising a CDR-1 comprising an amino acid sequence of SEQ ID NO. 46, a CDR-2 domain comprising an amino acid sequence of SEQ ID NO. 47, and a CDR-3 domain comprising an amino acid sequence of SEQ ID NO. 48; and (b) a light chain variable region comprising a CDR-1 domain comprising an amino acid sequence of SEQ ID NO. 49. a CDR-2 domain comprising an ammo acid sequence of SEQ ID NO. 50. and a CDR-3 domain comprising an amino acid sequence of SEQ ID NO. 45.

[0030] In some embodiments, the heavy chain variable region comprises an amino acid sequence at least 95% identical to SEQ ID NO. 51, and the light chain variable region comprises an amino acid sequence at least 95% identical to SEQ ID NO. 52. In certain embodiments, the heavy chain variable region comprises an amino acid sequence of SEQ ID NO. 51, and the light chain variable region comprises an amino acid sequence of SEQ ID NO. 52.

[0031] In some embodiments, the anti-matriptase-2 antibody or antigen-binding fragment thereof comprises: (a) heavy chain CDR-1, CDR-2, and CDR-3 domains contained within a heavy chain variable region comprising an amino acid sequence of SEQ ID NO. 51; and (b) light chain CDR-1, CDR-2, and CDR-3 domains contained within a light chain variable region comprising an amino acid sequence of SEQ ID NO. 52.

[0032] In some embodiments, the heavy chain comprises an amino acid sequence of SEQ ID NO. 53, and the light chain comprises an amino acid sequence of SEQ ID NO. 54.

[0033] In some embodiments, the method further comprises administering, or the use is in combination with, an antagonist of a transforming growth factor-beta (TGF-|3) superfamily ligand. In some embodiments, the antagonist is an activin II receptor ligand trap or is an activin MB receptor Fc fusion protein. In certain embodiments, the antagonist is luspatercept.

[0034] In some embodiments, the antagonist is an activin IIA receptor Fc fusion protein. In certain embodiments, the antagonist is sotatercept.BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG. 1 shows an illustration of the flow cytometry analysis methods used to track erythroid progenitor maturation, as described in the Example. FIG. 1 exhibits a representative plot showing LIN-Terl 19+erythroid progenitors. The assessment of maturation was based on forward light scatter (FSC, an indicator of cell size) and CD44 expression.

[0036] FIG. 2 shows results demonstrating that KYI 066 induced hepcidin expression and reduced iron overload in P-thalassemia intermedia (Hbbth3 / +) mice, as described in the Example. Panels A-D are plots of liver hepcidin (Hamp) mRNA expression levels (Panel A), serum hepcidin levels (Panel B), serum iron levels (Panel C). and liver iron content (Panel D) following treatment. Panel E shows representative images of Perl's Prussian blue-stained liver sections. Hbbl}'3' (8-week-old males) mice were injected every’ week with 10 mg / kg of KYI 066 for 8 weeks. Controls received 10 mg / kg of a non- relevant antibody of the same isotype (control IgGl). Hbb^3mice treated with RAP- 536L received 10 mg / kg of ActRIIB-mFc twice weekly for 8 weeks. Values in WT mice are shown for comparison. Results are presented by box plots with n=6-10 mice / group. Statistical significance was determined by Brown -Forsythe and Welch ANOVA tests followed by Tamhane's T2 multiple comparisons tests and is indicated by the brackets (* P<0.05; ** P<0.01; *** P<0.001).

[0037] FIG. 3 shows results of a combinatorial approach of KYI 066 with darbopoietin alpha, as described in the Example. Panels A-E are plots of liver hepcidin (Hamp) mRNA expression levels (Panel A), liver iron content (Panel B), hemoglobin levels (Panel C), hematocrit (Panel D), and red blood cells (Panel E) following treatment. Hbb& !+(8-week-old males) mice were injected every week with 10 mg / kg of KY1066 and 30mg / kg of darbopoietin alpha for 8 weeks. Controls received 10 mg / kg of a non-relevant antibody of the same isotype (control IgGl). Results are presented by box plots with n=6 / group. Statistical significance w as determined by student t test and is indicated by the brackets (* P<0.05; ** P<0.01).

[0038] FIG. 4 shows results demonstrating that KYI 066 ameliorates ineffective erythropoiesis in P-thalassemia intermedia (Hbb^3) mice, as described in the Example. Panels A and B shows results of flow cytometry’ analysis of the proportion of Teri 19+erythroid sub-populations in the spleen (Panel A) and the bone marrow (Panel B), usingTeri 19 and CD44 labelled antibodies. The additional parameter, FSC, was used to discriminate between five distinct stages of erythroid differentiation: (I) pro-erythroblasts, (II) basophilic, (III) polychromatic, (IV) orthochromatic cells, and reticulocytes and (V) red blood cells. Analysis was performed in control Hbb^^ mice and in Hbhlh3mice that received the different treatments (KY1066, RAP-536L, or the two combined). Data are presented by stacked bar graphs with mean ± SD of n=6-10 mice / group.

[0039] FIG. 5 shows results demonstrating that KYI 066 limits the formation of toxic a-chain / heme aggregates and reduces ROS formation in Hbb^3^ mice. Panel A shows TAU gel electrophoresis of membrane-bound globins in RBCs from WT mice, control Hbb&3J+mice, and Hbb&y+mice that received the different treatments (KYI 066, RAP- 536L, or the two combined). Panels B and C are plots of median fluorescence intensity of spleen (Panel B) and bone marrow (Panel C) Teri 19+erythroid cells generated following incubation with the ROS indicator CM-H2DCFDA. Panels D and E are plots of mean percentage of apoptotic (annexin V+) Teri 19+ery throid cells in the spleen (Panel D) and bone marrow (Panel E). Results are presented by box plots with n=6-10 mice / group. Statistical significance was determined by Brown-Forsythe and Welch ANOVA tests followed by7Tamhane's T2 multiple comparisons tests and is indicated by the brackets (* P<0.05; ** P<0.01; **** p<0.0001).

[0040] FIG. 6 show results demonstrating that RAP536L slightly reduces the formation of toxic a-chain / heme aggregates in Hbb'3"3' mice. TAU gel electrophoresis of membrane-bound globins in RBCs from control Hbb'i>3l+mice and Hbh'hi +mice that received RAP-536L is presented. Soluble a and (I Hb from WT RBCs are shown as a reference.

[0041] FIG. 7 shows results demonstrating that KYI 066 improves reticulocytosis and splenomegaly in Hbb&mice. Panels A-E are plots of reticulocyte count as a percentage of total red blood cells (Panel A), spleen index (Panel B), hemoglobin levels (Panel C), hematocrit (Panel D), and red blood cells (Panel E) in control Hbh'3' mice and Hbb&3 / +mice that received the different treatments (KY1066. RAP-536L, or the two combined). Results are presented by box plots with n=6-10 mice / group. Statistical significance was determined by Brown-Forsythe and Welch ANOVA tests followed by Tamhane's T2 multiple comparisons tests and is indicated by the brackets (** P<0.01;*** P<0.001: **** p<0.0001).DETAILED DESCRIPTION OF THE INVENTION

[0042] The practice of the present invention can employ, unless otherwise indicated, conventional techniques of pharmaceutics, formulation science, protein chemistry, cell biology7, cell culture, molecular biology, microbiology, recombinant DNA, immunology, clinical pharmacology, and clinical practice, which are within the skill of the art.

[0043] In order that the present invention can be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary7skill in the art to which this invention is related.

[0044] Any headings provided herein are not limitations of the various aspects or embodiments of the invention, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety7.

[0045] All references cited in this disclosure are hereby incorporated by reference in their entireties. In addition, any manufacturers’ instructions or catalogues for any products cited or mentioned herein are incorporated by reference. Documents incorporated by reference into this text, or any teachings therein, can be used in the practice of the present invention. Documents incorporated by reference into this text are not admitted to be prior art.Definitions

[0046] The phraseology or terminology in this disclosure is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0047] As used in this specification and the appended claims, the singular forms "‘a,” “an,” and “the” include plural referents, unless the context clearly dictates otherwise. The terms “a” (or “an”) as well as the terms “one or more” and “at least one” can be used interchangeably.

[0048] Furthermore, “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” asused in a phrase such as “A and / or B” is intended to include A and B, A or B, A (alone), and B (alone). Likewise, the term “and / or’ as used in a phrase such as “A, B, and / or C” is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).

[0049] Wherever embodiments are described with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of’ are included.

[0050] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range, and any individual value provided herein can serve as an endpoint for a range that includes other individual values provided herein. For example, a set of values such as 1, 2, 3, 8, 9, and 10 is also a disclosure of a range of numbers from 1-10, from 1-8, from 3-9, and so forth. Likewise, a disclosed range is a disclosure of each individual value (z.e., intermediate) encompassed by the range, including integers and fractions. For example, a stated range of 5-10 is also a disclosure of 5, 6, 7, 8. 9, and 10 individually, and of 5.2, 7.5, 8.7, and so forth.

[0051] Unless otherwise indicated, the terms “at least” or “about” preceding a series of elements is to be understood to refer to every element in the series. The term “about” preceding a numerical value includes ± 10% of the recited value. For example, a concentration of about 1 mg / mL includes 0.9 mg / mL to 1. 1 mg / mL. Likewise, a concentration range of about 1 % to 10% (w / v) includes 0.9% (w / v) to 11 % (w / v).

[0052] Amino acids are referred to herein by their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, are referred to by their commonly accepted single-letter codes.

[0053] The term “antibody” refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule. The terms “antibody” or “immunoglobulin” are used interchangeably herein.

[0054] A typical antibody comprises at least two heavy chains and two light chains interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chainvariable region and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI. CH2. and CH3. Each light chain is comprised of a light chain variable region and a light chain constant region (CL). The light chain constant region is comprised of one domain, Cl. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g, effector cells) and the first component (Clq) of the classical complement system.

[0055] Antibodies can be of any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, or subclasses (isotypes) thereof (e.g., IgGl. IgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. There are two classes of mammalian light chains, lambda and kappa.

[0056] The heavy and light chain variable regions can be further subdivided into regions of hypervariability, termed complementarity-determining regions (CDRs), interspersed with regions that are more conserved, termed framework (FW) regions. The CDRs in each chain are held together in close proximity' by the FW regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies. Each heavy and light chain variable region is composed of three CDRs and four FW regions, arranged from amino-terminus to carboxy-terminus in the following order: FW-1, CDR-1, FW-2, CDR-2, FW-3, CDR-3, FW-4.

[0057] There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (Kabat et al., 1991); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-Lazikani etal., 1997). In addition, combinations of these two approaches are sometimes used in the art to determine CDRs.

[0058] The amino acid position numbering as in Kabat, refers to the numbering system used for heavy chain variable domains or light chain variable domains (approximately residues 1-107 of the light chain and residues 1-1 13 of the heavy chain). Using this numbering system, the actual linear amino acid sequence can contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FW or CDRof the variable domain. For example, a heavy chain variable domain can include a single amino acid insert (residue 52a. according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc., according to Kabat) after heavy chain FW residue 82.

[0059] The Kabat numbering of residues can be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence. Chothia refers instead to the location of the structural loops (Chothia & Lesk, 1987). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software.

[0060] IMGT (ImMunoGeneTics) also provides a numbering system for the immunoglobulin variable regions, including the CDRs (see, e.g., Lefranc et al., 2003). The IMGT numbering system was based on an alignment of more than 5,000 sequences, structural data, and characterization of hypervariable loops and allows for easy comparison of the variable and CDR regions for all species. According to the IMGT numbering schema heavy chain variable region CDR-1 is at positions 26 to 35. heavy chain variable region CDR-2 is at positions 51 to 57, heavy chain variable region CDR-3 is at positions 93 to 102, light chain variable region CDR-1 is at positions 27 to 32, light chain variable region CDR-2 is at positions 50 to 52, and light chain variable region CDR-3 is at positions 89 to 97.

[0061] As used herein, the term “antibody” encompasses polyclonal antibodies; monoclonal antibodies; multispecific antibodies, such as bispecific antibodies generated from at least two intact antibodies; humanized antibodies; human antibodies; chimeric antibodies; fusion proteins comprising an antigen-determination portion of an antibody; and any other modified immunoglobulin molecule comprising an antigen recognition site, so long as the antibodies exhibit the desired biological activity.

[0062] A “monoclonal antibody” (mAb) refers to a homogeneous antibody population that is involved in the highly specific recognition and binding of a single antigenicdeterminant, or epitope. This is in contrast to polyclonal antibodies, which typically include different antibodies directed against different antigenic determinants. The term ■‘monoclonaf’ can apply to both intact and full-length monoclonal antibodies, as well as to antibody fragments (such as Fab, Fab’, F(ab’)2, Fv), single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. Furthermore, “monoclonal antibody” refers to such antibodies made in any number of ways including, but not limited to. by hybridoma, phage selection, recombinant expression, and transgenic animals.

[0063] The term “humanized antibody” refers to an antibody derived from a nonhuman ty.g, murine) immunoglobulin, which has been engineered to contain minimal non-human (e.g.. murine) sequences. Typically, humanized antibodies are human immunoglobulins in which residues from the CDR are replaced by residues from the CDR of a non-human species (e g, mouse, rat, rabbit, or hamster) that have the desired specificity', affinity, and capability (Jones et al., 1986; Riechmann et al., 1998; Verhoeyen et al.. 1988). In some instances, the Fv FW residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species that has the desired specificity, affinity, and capability.

[0064] Humanized antibodies can be further modified by the substitution of additional residues either in the Fv framework region and / or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and / or capability. In general, humanized antibodies will comprise substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDR regions that correspond to the non-human immunoglobulin whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. Humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Patent Nos. 5,225,539 and 5,639,641.

[0065] The term “human antibody” means an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any technique known in the art. The definition of a human antibody includes intact or full-length antibodies comprising at least one human heavy and / or lightchain polypeptide such as, for example, an antibody comprising murine light chain and human heavy chain polypeptides.

[0066] The term '‘antigen-binding fragment” refers to a portion of an intact antibody comprising the complementarity determining variable regions of the antibody. Examples of antibody fragments that can constitute an “antigen-binding fragment” include, but are not limited to, Fab. Fab’. F(ab‘)2, and Fv fragments, linear antibodies, single chain antibodies (e.g, ScFvs), and multi-specific antibodies formed from antibody fragments.

[0067] The terms “inhibit,” “block,” and “suppress” are used interchangeably and refer to any statistically significant decrease in a given biological activity7, including full blocking of the activity . For example, “inhibition” can refer to a decrease of about 10%, 20%. 30%, 40%. 50%. 60%. 70%. 80%. 90% or 100% in biological activity.Accordingly, when the terms “inhibition” or “suppression” are applied to describe, e.g., an effect of an anti-matriptase-2 antibody or antigen-binding fragment thereof, the terms may refer to the ability of an anti-matriptase-2 antibody or antigen-binding fragment thereof to statistically significantly increase: (a) bone morphogenetic protein (BMP) / sons of mothers against decapentaplegic homologue protein (SMAD) signaling, or (b) cleavage by matriptase-2 of a matriptase-2 substrate such as BMP co-receptor, or (c) hepcidin expression, and the like. Inhibition may be determined relative to an untreated control — for example, a control not treated with the anti-matriptase-2 antibody or antigenbinding fragment thereof. In some embodiments, an anti-matriptase-2 antibody or antigen-binding fragment thereof can inhibit an activity of matriptase-2 (such as those listed above) by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% or about 100%, as determined, for example, by flow cytometry, Western blotting, ELISA, proliferation assays, or other assays known to those of skill in the art.

[0068] By “subject” or “individual” or “animal” or “patient” or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, sports animals, and zoo animals including, e.g., humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, and so on.

[0069] The term “pharmaceutical composition” refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective and whichcontains no additional components that are unacceptably toxic to a subject to which the composition would be administered. Such composition can be sterile and can comprise a pharmaceutically acceptable carrier, such as physiological saline. Suitable pharmaceutical compositions can comprise one or more of a buffer (e.g, acetate, phosphate or citrate buffer), a surfactant (e.g., polysorbate), a stabilizing agent (e.g, human albumin), a preservative (e.g., benzyl alcohol), an absorption promoter to enhance bioavailability and / or other conventional solubilizing or dispersing agents.

[0070] An “effective amount” of an antibody or antigen-binding fragment thereof as disclosed herein is an amount sufficient to carry7out a specifically stated purpose. An “effective amount” can be determined empirically and in a routine manner, in relation to the stated purpose.

[0071] The anti-matriptase-2 antibody or antigen-binding fragment thereof of the invention can be naked or conjugated to other molecules such as toxins, labels, etc. The term “label” when used herein refers to a detectable compound or composition that is conjugated directly or indirectly to an antibody or antigen-binding fragment thereof, so as to generate a “labeled” antibody or antigen-binding fragment thereof. The label can be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, as in the case of, for instance, an enzy matic label, can catalyze chemical alteration of a substrate compound or composition that is detectable.

[0072] Terms such as “treating” or “treatment” or “to treat” or “alleviating” or “to alleviate” refer to therapeutic measures that cure, slow down, lessen symptoms of, and / or halt progression of a diagnosed pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder. In certain embodiments, a subject is successfully “treated” for a disease or disorder according to the methods provided herein if the patient shows, e.g., total, partial, or transient lessening or elimination of symptoms associated with the disease or disorder.

[0073] Terms such as “alleviating” or “to alleviate” refer to therapeutic measures that lessen the severity of a diagnosed pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder.

[0074] “Prevent” or “prevention” refer to prophylactic or preventative measures that prevent and / or slow the development or recurrence of a targeted pathologic condition or disorder. Thus, those in need of prevention include those prone to have or susceptible tothe disorder, including those who have had the disorder and are susceptible to recurrence. In certain embodiments, a disease or disorder is successfully prevented according to the methods provided herein if the patient develops, transiently or permanently, e.g., fewer or less severe symptoms or pathology' associated with the disease or disorder, or a later onset of symptoms or pathology associated with the disease or disorder, than a patient who has not been subject to the methods of the invention.

[0075] The terms “polypeptide,” “peptide.” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids and non-amino acids can interrupt it. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. In certain embodiments, the polypeptides can occur as single chains or associated chains.

[0076] A “conservative amino acid substitution” is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g, lysine, arginine, histidine), acidic side chains (e.g, aspartic acid, glutamic acid), uncharged polar side chains (e.g, asparagine, glutamine, serine, threonine, ty rosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, try ptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. In certain embodiments, conservative substitutions in the amino acid sequences of the antibody or antigen-binding fragment thereof of the invention do not abrogate the binding of the antibody or antigen-binding fragment thereof to the antigen(s). i.e.. matriptase-2, to which the antibody or antigen-binding fragment thereof binds. Methods of identifying conservative nucleotide and amino acid substitutions which do not eliminate antigen-binding are well-known in the art (see, e.g., Brummell et al., 1993; Kobayashi et al., 1999; Burks et al., 1997).

[0077] A “polynucleotide,'’ as used herein can include one or more “nucleic acids,"’ “nucleic acid molecules,” or “nucleic acid sequences,” and refers to a polymer of nucleotides of any length, and includes DNA and RNA. The polynucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogs. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[0078] The term “vector"’ means a construct, which is capable of delivering and, in some embodiments expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[0079] An “isolated” polypeptide, antibody, antigen-binding fragment, polynucleotide, vector, or cell is in a form not found in nature. Isolated polypeptides, antibodies, antigen-binding fragments, polynucleotides, vectors, or cells include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some embodiments, a polypeptide, antibody, antigen-binding fragment, polynucleotide, vector, or cell that is isolated is substantially pure. When used herein, the term “substantially pure” refers to purity of greater than 75%, preferably greater than 80% or 90%, and most preferably greater than 95%.

[0080] The terms “identical” or percent “identity” in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences.

[0081] One such non-limiting example of a sequence alignment algorithm is the algorithm described in Karlin & Altschul (1990), as modified in Karlin el al. (1993), and incorporated into the NBLAST and XBLAST programs (Altschul el al., 1997). In certain embodiments, Gapped BLAST can be used as described in Altschul et al. (1997). BLAST-2, WU-BLAST-2 (Altschul & Gish, 1996), ALIGN, ALIGN-2 (Genentech, South San Francisco. California) or Megalign (DNASTAR) are additional publicly available software programs that can be used to align sequences. In certain embodiments, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (e.g. , using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 90 and a length weight of 1, 2, 3, 4, 5, or 6). In certain alternative embodiments, the GAP program in the GCG software package, which incorporates the algorithm of Needleman and Wunsch (1970), can be used to determine the percent identity between two amino acid sequences (e.g., using either a BLOSUM 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5). Alternatively, in certain embodiments, the percent identity between nucleotide or amino acid sequences is determined using the algorithm of Myers and Miller (CABIOS 4: 11-17 (1989)). For example, the percent identity can be determined using the ALIGN program (version 2.0) and using a PAM120 with residue table, a gap length penalty of 12 and a gap penalty of 4. One skilled in the art can determine appropriate parameters for maximal alignment by particular alignment software. In certain embodiments, the default parameters of the alignment software are used.

[0082] In certain embodiments, the percentage identity “X’" of a first amino acid sequence to a second sequence amino acid is calculated as 100 x (Y / Z). where Y is the number of amino acid residues scored as identical matches in the alignment of the first and second sequences (as aligned by visual inspection or a particular sequence alignment program) and Z is the total number of residues in the second sequence. If the length of a first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence.Anti-Matriptase Antibodies and Antigen-Binding Fragments Thereof

[0083] Matriptase-2 is a type-II transmembrane trypsin-like serine protease encoded by the gene Tmprss6. It is mainly expressed by hepatocytes and controls hepcidin expression through the suppression of BMP / SMAD signaling by potentially cleaving BMP co-receptors such as hemojuvelin (Knutson, 2009; Stimberg & Gtitschow, 2013).

[0084] The matriptase-2 protein, and the nucleotide sequences that encode it are well known in the art, and both the nucleotide and amino acid sequences of matriptase-2 from several different species (including humans and mice) are publicly available, for example in the GenBank / NCBI database.

[0085] Exemplary anti-matriptase-2 antibodies of the present invention include the antibodies referred to herein as “NORI-003” (also referred to herein as “KYI 066”), “NORI-006,” “NORI-008,” and “NORI-011,” and antigen binding fragments thereof, such as antigen binding fragments that comprise the CDRs of these antibodies. The amino acid sequences for the CDRs, heavy chain and light chain variable regions, and full heavy and light chains ofNORI-003, NORI-006, NORI-008, and NORI-011, are provided in Tables 1-4, respectively, which also provides SEQ ID NOs for each amino acid sequence.Table 1. Amino acid sequences of NORI-003 CDRs (according to IMGT and Kabat numbering), heavy and light chain variable regions, and full heavy and light chains.Table 2. Amino acid sequences of NORI-006 CDRs (according to IMGT and Kabat numbering), heavy and light chain variable regions, and full heavy and light chains.Table 3. Amino acid sequences of NORI-008 CDRs (according to IMGT and Kabat numbering), heavy and light chain variable regions, and full heavy and light chains.Table 4. Amino acid sequences of NORI-OOl 1 CDRs (according to IMGT and Kabat numbering), heavy and light chain variable regions, and full heavy and light chains.

[0086] In addition to providing the specific anti-matriptase-2 antibodies, and fragments thereof, whose sequences are provided in Tables 1-4 above, the presentinvention also encompasses variants and equivalents of these anti-matriptase-2 antibodies and antibody fragments. For example, in some embodiments variants of the specific sequences disclosed herein that comprise one or more substitutions, additions, deletions, or other mutations may be used. A heavy chain variable region and / or light chain variable region amino acid sequence or portion thereof can be, e.g., 85%, 90%, 95%, 96%, 97%, 98% or 99% similar to a sequence set forth herein, and / or comprise 1, 2, 3, 4, 5 or more substitutions, e.g.. conservative substitutions, relative to a sequence set forth herein. In some embodiments an anti-matriptase-2 antibody or fragment thereof comprising a heavy chain variable region and / or light chain variable region amino acid sequence, or portion thereof, that is 85%, 90%, 95%, 96%, 97%, 98% or 99% similar to that present in the specific sequences provided herein (e.g., SEQ ID NO. 12, 13, 22, 23, 36, 37, 51, and / or 52) set forth herein, and / or comprise 1, 2, 3, 4, 5 or more substitutions, e.g., conservative substitutions, relative to that sequence, but comprises the specific CDR sequences found within such heavy chain and / or light chain variable regions — i. e. , any mutations (such as substitutions, additions, deletions, etc.) are outside of the CDRs.

[0087] Subsequent sections of this patent disclosure provide further details regarding different variants of the specific anti-matriptase-2 antibodies and fragments thereof described herein that are within the scope of the present invention, and how to make and use such variants.

[0088] In some embodiments, the anti-matriptase-2 antibody is a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a bi-specific antibody, a multispecific antibody, or any combination thereof. In some embodiments, anti-matriptase-2 antibody comprises a Fab, a Fab?, a F(ab’)2. a Fd, a Fv, a scFv, a disulfide linked Fv. a V-NAR domain, an IgNar. an intrabody, an IgGACH2, a minibody, a F(ab’)s a tetrabody, a triabody, a diabody, a single-domain antibody, DVD-Ig, Fcab, mAb2, a (scFv)2, or a scFv-Fc.

[0089] An anti-matriptase-2 antibody or antigen-binding fragment thereof provided herein can include, in addition to a heavy chain variable region and a light chain variable region, a heavy chain constant region or fragment thereof. In certain embodiments the heavy chain constant region is a human heavy chain constant region, e.g., a human IgG constant region, e.g.. a human IgGl constant region.

[0090] In certain embodiments, anti-matriptase-2 antibodies or antigen-binding fragments thereof of the invention are produced to comprise an altered Fc region, in which one or more alterations have been made in the Fc region in order to change functional and / or pharmacokinetic properties of the antibody or antigen-binding fragment thereof. Such alterations may result in altered effector function, reduced immunogenicity, and / or an increased serum half-life. The Fc region interacts with a number of ligands, including Fc receptors, the complement protein Clq. and other molecules, such as proteins A and G. These interactions are essential for a variety of effector functions and downstream signaling events including antibody dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC).

[0091] In some embodiments, a heavy chain constant region or fragment thereof can include one or more amino acid substitutions relative to a wild-type IgG constant domain, wherein the modified IgG has an increased half-life compared to the half-life of an IgG having the wild-type IgG constant domain. For example, the IgG constant domain can contain one or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, wherein the amino acid position numbering is according to the EU index as set forth in Kabat. In certain embodiments the IgG constant domain can contain one or more of a substitution of the amino acid at Kabat position 252 with Tyrosine (Y), Phenylalanine (F), Tryptophan (W), or Threonine (T). a substitution of the amino acid at Kabat position 254 with Threonine (T), a substitution of the amino acid at Kabat position 256 with Serine (S), Arginine (R), Glutamine (Q), Glutamic acid (E), Aspartic acid (D), or Threonine (T), a substitution of the amino acid at Kabat position 257 with Leucine (L), a substitution of the amino acid at Kabat position 309 with Proline (P). a substitution of the amino acid at Kabat position 311 with Serine (S), a substitution of the amino acid at Kabat position 428 with Threonine (T), Leucine (L), Phenylalanine (F), or Serine (S), a substitution of the amino acid at Kabat position 433 with Arginine (R), Serine (S), Isoleucine (I), Proline (P), or Glutamine (Q), or a substitution of the amino acid at Kabat position 434 with Tryptophan (W), Methionine (M), Serine (S), Histidine (H), Phenylalanine (F), or Tyrosine. More specifically, the IgG constant domain can contain amino acid substitutions relative to a wild-type human IgG constant domain including as substitution of the amino acid at Kabat position 252 with Tyrosine (Y), asubstitution of the amino acid at Kabat position 254 with Threonine (T), and a substitution of the amino acid at Kabat position 256 with Glutamic acid (E).

[0092] An anti-matriptase-2 antibody or antigen-binding fragment thereof provided herein can include a light chain constant region or fragment thereof. In certain embodiments the light chain constant region is a kappa constant region or a lambda constant region, e.g., a human kappa constant region or a human lambda constant region.

[0093] In some embodiments, the disclosure provides a composition, e.g., a pharmaceutical composition, comprising an anti-matriptase-2 antibody or antigen-binding fragment thereof of the invention, optionally further comprising one or more carriers, diluents, excipients, or other additives. Preferably, the composition comprises one or more carriers, diluents, excipients, or other additives. For example, the composition can comprise one or more bulking agents (e.g., dextran 40, glycine, lactose, mannitol, trehalose), one or more buffers (e.g., acetate, citrate, histidine, lactate, phosphate, Tris), one or more pH adjusting agents (e.g, hydrochloric acid, acetic acid, nitric acid, potassium hydroxide, sodium hydroxide), and / or one or more diluents (e.g., water, physiological saline). The pH of the composition is preferably between about 3.0 and 8.0. In one embodiment, the pH is between about 3.5 and 6.5, or between about 5.0 and 7.5.Uses of Anti-Matriptase-2 Antibody or Antigen-Binding Fragment Thereof

[0094] The present invention provides various methods of using the anti-matriptase-2 antibody or antigen-binding fragment thereof described herein. Such methods include, but are not limited to, use of the anti-matriptase-2 antibody or antigen-binding fragment thereof described herein for treating one or more pathologies associated with (3- thalassemia in a subject in need thereof, and for alleviating one or more pathologies associated with (3-thalassemia in a subject in need thereof. These methods may comprise administering to the subject a pharmaceutical composition comprising an effective amount of the anti-matriptase-2 antibody or antigen-binding fragment thereof.

[0095] The one or more pathologies may be selected from insoluble liver iron stores; a-globin precipitates in erythroid cells; ROS formation; apoptosis of ery throid cells; reticulocytosis; and a combination thereof. Thus, in some embodiments, the present invention relates to a method for treating or alleviating insoluble liver iron stores in a subject with P-thalassemia; a method for treating or alleviating a-globin precipitates inerythroid cells of a subject with p-thalassemia; a method for treating or alleviating ROS formation in a subject with p-thalassemia; a method for treating or alleviating apoptosis of erythroid cells in a subject with P-thalassemia; and / or a method for treating or alleviating reticulocytosis in a subject with P-thalassemia. These methods may comprise administering to the subject a pharmaceutical composition comprising an effective amount of the anti-matriptase-2 antibody or antigen-binding fragment thereof.

[0096] In some embodiments, the a-globin may precipitate in erythrocytes. In some embodiments, the ROS formation may occur in the spleen, bone marrow, or both. Further, in some embodiments, the apoptosis many occur in ery throid cells in the spleen, bone marrow, or both.

[0097] The efficacy of a given composition or method of use can be demonstrated or assessed using standard methods known in the art, such as methods that compare the efficacy of a given / “test” composition or method to a “control” composition or method. For example, the efficacy of a given composition or method in treating or alleviating a pathology’ associated with p-thalassemia may be demonstrated or assessed by comparing its ability to improve one or more indicators of the pathology as compared to that of a control composition or control method, such as a placebo control. For instance, a comparison can be made between different subjects (e.g, between a test group of subjects or a control group of subjects). Similarly, the efficacy of a given composition or method in treatment can be demonstrated or assessed in a single subject by comparing the pathology before and after treatment.

[0098] Methods of preparing the pharmaceutical composition for administration to a subject, and methods of administering the pharmaceutical composition to a subject, are well-known to those of ordinary skill in the art, or can be readily determined by those of ordinary skill in the art. For example, the route of administration of the pharmaceutical composition can be, for example, oral, parenteral, by inhalation, or topical. The term “parenteral” as used herein includes, e.g., intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, and vaginal administration. Oral dosage forms include, e.g.. capsules, tablets, aqueous suspensions, and solutions. Nasal aerosol or inhalation dosage forms can be prepared, for example, as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, and / or other conventional solubilizing or dispersing agents.

[0099] Usually, a suitable pharmaceutical composition can comprise a buffer (e.g, acetate, phosphate or citrate buffer), optionally a surfactant (e.g, polysorbate), optionally a stabilizer agent (e.g., human albumin), etc. The form and character of the pharmaceutically acceptable carrier or diluent can be dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well- known variables.

[0100] As discussed herein, the anti-matriptase-2 antibody or antigen-binding fragment thereof can be administered in a therapeutically effective amount for the uses described herein. In this regard, it will be appreciated that the disclosed anti-matriptase-2 antibody or antigen-binding fragment thereof can be formulated to facilitate administration and promote stability of the anti-matriptase-2 antibody or antigen-binding fragment thereof. Pharmaceutical compositions in accordance with the present invention can comprise a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, non-toxic buffers, preservatives and the like. For the purposes of the instant application, a “therapeutically effective amount"' of an anti-matriptase-2 antibody or antigen-binding fragment thereof means an amount sufficient to achieve a benefit, e.g., to ameliorate symptoms of a disease or condition (e.g., one or more pathologies of [3- thalassemia). Suitable formulations for use in the therapeutic methods disclosed herein are described in Remington's Pharmaceutical Sciences (2000).

[0101] The composition can be administered as a single dose, multiple doses, or over an established period of time in an infusion. Dosage regimens also can be adjusted to provide the optimum desired response (e.g. , a therapeutic or prophylactic response). The amount of an anti-matriptase-2 antibody or antigen-binding fragment thereof that can be combined with carrier materials to produce a dosage form will vary depending upon many different factors, including means of administration, target site, physiological state of the patient (i.e., the severity of the disease, the history of the disease, and the age, height, weight, health, and physical condition of the individual undergoing therapy), whether treatment is prophylactic or therapeutic, other medications administered, and whether the subject is a human or an animal. Usually, the subject is a human, but nonhuman mammals, including transgenic mammals, can also be treated. The amount of an anti-matriptase-2 antibody or antigen-binding fragment thereof to be administered is readily determined by one of ordinary skill in the art without undue experimentation,given this disclosure. Treatment dosages can be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.

[0102] The anti-matriptase-2 antibody or antigen-binding fragment thereof of the invention can be administered in combination with one or more additional active agents. In some embodiments, the additional active agent may be an antagonist of a TGF-p superfamily ligand, such as an activin II receptor ligand trap. For example, the antagonist may be an activin MB receptor Fc fusion protein such as luspatercept, or an activin II A receptor Fc fusion protein such as sotatercept. In other embodiments, the active agent may be an erythropoiesis enhancing agents such as darbopoietin alpha.

[0103] In embodiments in which more than one active agent is administered, the agents can be administered together (for example, in the same formulation and / or at the same time), or separately (for example, in different formulations and / or at different times). In some such embodiments, the agents are administered systemically. In some such embodiments, the agents are administered locally. In some such embodiments, one (or more) agent is administered systemically, and one (or more) agent is administered locally. Where two such agents are used, it may be possible to use lower dosages or amounts of each agent, as compared to the dosages necessary7when each agent is used alone.

[0104] This disclosure also provides for the use of an anti-matriptase-2 antibody or antigen-binding fragment thereof as described herein in the manufacture of a medicament for treating or alleviating one or more pathologies associated with |3-thalassemia.Methods of Preparation

[0105] The anti-matriptase-2 antibody or antigen-binding fragment thereof can be prepared using hybridoma methods, such as those described by Kohler & Milstein (1975). Using the hybridoma method, a mouse, hamster, or other appropriate host animal, is immunized to elicit the production by lymphocytes of antibodies that will specifically bind to an immunizing antigen. Lymphocytes can also be immunized in vitro. Following immunization, the lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol (PEG), to form hybridoma cells that can then be selected away from unfused lymphocytes and myeloma cells. Hybridomas that produce monoclonal antibodies directed specifically against a chosen antigen as determined byimmunoprecipitation, immunoblotting, or by an in vitro binding assay (e.g., RIA or ELISA) can then be propagated either in in vitro culture using standard methods (Goding, 1986).

[0106] The anti-matriptase-2 antibody or antigen-binding fragment thereof can also be made using recombinant DNA methods, for example, as described in U.S. Patent No. 4,816,567. In some instances, the polynucleotides encoding a monoclonal antibody are isolated from mature B-cells or hybridoma cell, such as by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequence is determined using conventional procedures. The isolated polynucleotides encoding the heavy and light chains or antigen-binding fragments thereof are then cloned into suitable expression vectors, which when transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, antibodies or antigenbinding fragments thereof are generated by the host cells. Also, the anti-matriptase-2 antibody or antigen-binding fragment thereof can be isolated from phage display libraries expressing CDRs of the desired species, as described by McCafferty et al. (1990), Clackson et al. (1991), and Marks et al. (1991).

[0107] The polynucleotide(s) encoding an anti-matriptase-2 antibody or antigenbinding fragment thereof can further be modified in a number of different manners using recombinant DNA technology to generate alternative antibodies or antigen-binding fragments thereof. In some embodiments, the constant domains of the light and heavy chains of, for example, a mouse monoclonal antibody can be substituted (1) for those regions of, for example, a human antibody to generate a chimeric antibody or (2) for a non-immunoglob ulin polypeptide to generate a fusion antibody. In some embodiments, the constant regions are truncated or removed to generate the desired antibody fragment of a monoclonal antibody. Site-directed or high-density mutagenesis of the variable region can be used to optimize specificity, affinity, etc. of a monoclonal antibody.

[0108] In certain embodiments, the anti-matriptase-2 antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof. Human antibodies can be directly prepared using various techniques known in the art. Immortalized human B lymphocytes immunized in vitro or isolated from an immunizedindividual that produce an antibody directed against a target antigen can be generated (see, e.g.. Cole et al.. 1985; Boemer et a / .. 1991; and U.S. Patent No. 5.750,373).

[0109] The anti-matriptase-2 antibody or antigen-binding fragment thereof can be selected from a phage library, where the phage library expresses human antibodies, as described, for example, by Vaughan et al. (1996), Sheets et al. (1998), and Marks et al. (1991). Techniques for the generation and use of antibody phage libraries are also descnbed in U.S. Patent Nos. 5,969.108. 6,172,197. 5,885,793. 6.521,404; 6.544.731; 6,555,313; 6,582,915; 6,593,081; 6,300,064; 6,653,068; 6,706,484; and 7,264,963; and in Rothe et al. (2007).

[0110] Affinity maturation strategies and chain shuffling strategies are known in the art and can be employed to generate high affinity human antibodies or antigen-binding fragments thereof (see Marks et al., 1992).

[0111] In some embodiments, the anti-matriptase-2 antibody or antigen-binding fragment thereof can be a humanized antibody or antigen-binding fragment thereof. Methods for engineering, humanizing, or resurfacing non-human or human antibodies can also be used and are well known in the art. A humanized, resurfaced, or similarly engineered antibody can have one or more amino acid residues from a source that is non- human, e.g., mouse, rat, rabbit, non-human primate, or other mammal. These non-human amino acid residues are replaced by residues that are often referred to as ‘‘import'’ residues, which are typically taken from an “import” variable, constant, or other domain of a known human sequence. Such imported sequences can be used to reduce immunogenicity or reduce, enhance, or modify binding, affinity', on-rate, off-rate, avidity’, specificity', half-life, or any other suitable characteristic, as known in the art. In general, the CDR residues are directly and most substantially involved in influencing binding.Accordingly, part or all of the non-human or human CDR sequences are maintained while the non-human sequences of the variable and constant regions can be replaced with human or other amino acids. Humanization, resurfacing, or engineering of anti- matriptase-2 antibodies or antigen-binding fragments thereof can be performed using any known method, such as, but not limited to, those described in, Jones et al. (1986);Riechmann et al. (1988); Verhoeyen et al. (1988); Sims et al. (1993); Chothia & Lesk (1987); Carter et al. (1992); Presta ef uZ. (1993); U.S. Patent Nos. 5,639,641, 5,723,323; 5,976,862; 5,824,514; 5,817.483; 5,814,476; 5,763,192; 5,723,323; 5,766,886; 5.714,352;6,204,023; 6,180,370; 5,693.762; 5,530,101; 5,585,089: 5,225,539; 4,816,567, 7,557,189; 7,538,195; and 7,342,110; International Application Nos. PCT / US98 / 16280; PCT / US96 / 18978; PCT / US91 / 09630; PCT / US91 / 05939; PCT / US94 / 01234;PCT / GB89 / 01334; PCT / GB91 / 01134; PCT / GB92 / 01755; International Patent Application Publication Nos. WO90 / 14443; WO90 / 14424; W090 / 14430; and European Patent Publication No. EP 229246.

[0112] Anti-matriptase-2 humanized antibodies and antigen-binding fragments thereof can also be made in transgenic mice containing human immunoglobulin loci that are capable, upon immunization, of producing the full repertoire of human antibodies in the absence of endogenous immunoglobulin production. This approach is described in U.S. Patent Nos. 5.545,807; 5,545,806; 5,569.825; 5,625,126: 5,633,425; and 5,661,016.

[0113] V arious techniques are known for the production of antibody fragments. Traditionally, these fragments are derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto & Inouye (1993); Brennan et al. (1985)). In certain embodiments, anti-matriptase-2 antibody fragments are produced recombinantly. Fab, Fv, and scFv antibody fragments can all be expressed in and secreted from E. coli or other host cells, thus allowing the production of large amounts of these fragments. Such anti-matriptase-2 antibody fragments can also be isolated from the antibody phage libraries discussed above. Anti-matriptase-2 antibody fragments can also be linear antibodies, as described in U.S. Patent No. 5.641.870. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner.

[0114] According to the present invention, techniques can be adapted for the production of single-chain antibodies specific to matripase-2 (see, e.g., U.S. Patent No. 4,946,778). In addition, methods can be adapted for the construction of Fab expression libraries (see, e.g, Huse et al., 1989) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for matiptase-2. Antibody fragments can also be produced by techniques in the art including, but not limited to: (a) a F(ab')2 fragment produced by pepsin digestion of an antibody molecule; (b) a Fab fragment generated by reducing the disulfide bridges of an F(ab’)2 fragment, (c) a Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent, and (d) Fv fragments.

[0115] An anti-matriptase-2 antibody or antigen-binding fragment thereof can be modified to contain additional chemical moieties not normally part of the protein. Such moieties can improve the characteristics of the antibody or antigen-binding fragment thereof for example, solubility’, biological half-life, or absorption. The moieties can also reduce or eliminate any undesirable side effects of the antibody or antigen-binding fragment thereof. An overview of those moieties can be found in Remington's Pharmaceutical Sciences (2000).

[0116] In particular embodiments, anti-matriptase-2 antibodies or antigen-binding fragments thereof may be prepared by methods described in PCT Publication No. WO 2021 / 105389, which is incorporated herein by reference.EXAMPLES

[0117] Embodiments of the present disclosure can be further defined by reference to the following non-limiting example. It will be apparent to those skilled in the art that many modifications, both to materials and methods, can be practiced without departing from the scope of the present disclosure.

[0118] A study was conducted examining the effects of a fully human monoclonal antibody neutralizing matriptase-2 on the P-thalassemia intermedia phenoty pe of mice with heterozygous deletion of the (3-minor and 0-major hemoglobin genes (Hbbth3 / +). These mice have moderately severe, transfusion-independent, hypochromic microcytic anemia associated with ineffective erythropoiesis resulting in splenomegaly, hepcidin suppression, and secondary7iron overload. The matriptase-2 neutralizing antibody was also administered in combination with a RAP-536 like molecule (RAP-536L), the murine version of Luspatercept (Cappellini et al., 2020), and the benefits of using this drug combination for Hbb^l+mice were tested. In addition, the matriptase-2 neutralizing antibody was administered in combination with darbopoetin alpha since agents enhancing erythropoiesis could increase the production of red blood cells and could have a synergic effect with the inhibition of matriptase-2.MethodsMatriptase-2 neutralizing antibody KY1066

[0119] Transgenic mice genetically engineered to contain the entire human immunoglobulin heavy and light chain variable-gene repertoire (Lee et al., 2014) wereimmunized with purified recombinant human and mouse matriptase-2 extra-cellular domains, full-length human matriptase-2 expressed on cells, and matriptase-2 encoding DNA vectors, generating a diverse range of highly specific monoclonal antibodies. KYI 066 was identified after a multi-step screening cascade on the Kymab IntelliSelect platform (www.kymab.com) as a cross-reactive antibody (human / mouse) blocking the protease activity of matriptase-2 by binding to the serine protease active site. It was shown to increase Bmp / Smad signaling and hepcidin expression in C57B1 / 6 mice, following a single IP dose of 10 mg / kg (Wake et al., 2019). For long-term assessment in Hbb^' mice, KYI 066 was reformatted into a mouse IgGl chimeric antibody reduce the formation an anti-drug-antibodies (ADA) response.Animal Studies

[0120] (3-thalassemic (Hob6''' ) mice and wild-type (WT) controls used in this study were all 8-week-old males on a C57BL / 6N background. Mice were housed under a constant light-dark cycle. They were given free access to tap water and standard laboratory mouse diet containing 180 parts per million (ppm) iron (SSNIFF, Soest, Germany). Mbh1^ ' mice were treated with intra-peritoneal injections (10 mg / kg) of the anti-matriptase-2 antibody KYI 066 or anon-relevant mouse IgGl isotype control once a week for 8 weeks. RAP-536L, a modified activin receptor type IIB extracellular domain linked to the murine IgG2a-Fc domain biochemically identical to RAP-536 (Suragani et al.. 2014a). was administered by intraperitoneal injections at the dose of 10 mg / kg twice weekly for 8 weeks. Mice received intraperitoneal injections (30 mg / kg) of darbopoietin alpha (Arasnep, Amgan) once a week. The mice were humanely killed 3 days after the last injection.Gene Expression Analysis

[0121] Total RNA from mouse liver was extracted using UPzol lysis reagent (biotechrabbit, Hennigsdorf, Germany). cDNA was synthesized using MMLV-RT (Promega). Quantitative PCR (Q-PCR) reactions with Hamp and Hprt primers listed in Belot et al. (2020) were prepared with LightCycler® 480 DNA SYBR Green I Master reaction mix (Roche Diagnostics) and run on a LightCycler® 480 System (Roche Diagnostics). ACt values were obtained by subtracting the reference gene Ct to the target gene Ct.Serum Hepcidin Assay

[0122] Serum hepcidin was quantified using the Hepcidin Murine Compte ELISA kit (Intrinsic Lifesciences, La Jolla, CA, USA) according to manufacturer’s instructions.Hematological and iron parameters

[0123] Blood was collected at sacrifice from the abdominal aorta in EDTA-coated tubes. Hematological parameters were assessed on a CELL-DYN Emerald system (Abbott, Lake Forest, IL. USA). Serum iron concentration was measured by the Anexplo core facility of Toulouse. The Reticulocyte Reagent System (BD 349204) was used to determine reticulocytes as a percentage of total erythrocytes in the peripheral blood.Tissue iron measurements and iron staining

[0124] Quantitative measurement of non-heme iron in the liver was performed according to the method recommended by Torrance & Bothwell (1980). Liver samples were fixed in 4% buffered formalin and embedded in paraffin. Deparaffinized tissue sections were stained with the Peris’ Prussian blue stain for non-heme iron and counterstained with nuclear fast red. Slides were scanned on a Pannoramic 250 Flash II (3DHISTECH) and analyzed with the Pannoramic Viewer software.Erythroid differentiation in bone marrow and spleen

[0125] Ery throid cells were analyzed from the spleen and the bone marrow by flow cytometry. Cell suspensions were stained in PBS supplemented with sterile 5% FBS. The following monoclonal antibodies were used for murine flow cytometric analysis: anti-Terl 19-PE (BD biosciences, 553673), anti-CD44-PE-Cy7 (BD biosciences, 560599), lineage (Lin) APC-conjugated antibodies: anti-Ly-6G (BD biosciences, 560599), anti- Cdl lc (BD biosciences, 561119), anti-CD3 (BD biosciences, 565643), anti-cdl lb (BD biosciences, 553312), anti-CD49b (BD biosciences. 560628), anti-CD19 (BD biosciences, 550992). Specifically, erythrocytes were defined as Lin-, Terl l9+. This assay allows the separation of erythroid cells into distinct populations corresponding to (I) pro-ery throblasts, (II) basophilic, (III) polychromatic, (IV) orthochromatic cells and reticulocytes, and (V) red blood cells (FIG. 1). A minimum of 250,000 events were recorded for erythrocytes in spleen and bone marrow. For all the analyses, cells were acquired using the MACSQuant® Analyzer 10 Flow Cytometer (Miltenyi Biotec) and the results analyzed with FlowJo software (Tree Star Inc.).Detection of membrane a-globins in erythroid cells

[0126] Blood samples were collected, and membrane cytoskeletons were prepared as previously described (Sorensen et al., 1990) from the same number of erythrocytes for each sample (150xl06). Briefly, the erythrocyte ghosts were washed three times by resuspending in hypotonic lysis buffer and centrifugation at 21,000x g. Membrane lipids were extracted in 50 mM sodium borate pH 8. ImM EDTA, 0.5% Triton X-100 (Sigma- Aldrich) and protease inhibitors. After a last 30 min centrifugation at 30 OOOxg the supernatant was completely removed and the triton-insoluble pellet corresponding to the membrane cytoskeletons was snap frozen and analyzed using Triton / acetic acid / urea (TAU) polyacrylamide gel electrophoresis, as previously described (Alter, 1981).Reactive oxygen species measurement

[0127] Cells from the spleen and bone marrow were washed and re-suspended in PBS supplemented with 5% fetal bovine serum and incubated with 5-(and -6)-choloromethyl- 2’,7’-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) stain (Invitrogen, C6827) (5 pmol / 1) in the dark for 15 min at 37° C. centrifugated 5 min at 300 g and resuspended in PBS 5% FBS. The oxidative conversion of CM-H2DCFDA to its fluorescent product by ROS was measured immediately by flow cytometry. The ROS fluorescence signals (median fluorescence intensity) were recorded in the Lin- Teri 19+cell population. For all the analyses, cells were acquired using the MACSQuant® Analyzer 10 Flow Cytometer and the results analyzed with FlowJo software (Tree Star Inc., Ashland, OR USA).Apoptosis assessment

[0128] Apoptosis in erythroid precursors was determined by flow cytometry using an apoptosis detection kit (BD Biosciences). Erythroid precursors in spleen and bone marrow were identified as previously described and counter stained with annexin V labeled with Pacific blue (Biolegend, 640918). The percentages of annexin V+ among Ter-119+cells were gated, and the data recorded.Statistical analysis

[0129] Experimental data are presented as box plots or stacked bar charts. One-way analysis of variance (ANOVA) with Brown-Forsythe and Welch tests, followed byTamhane's T2 multiple comparisons tests, was performed using GraphPad Prism Version 9.3.0 (GraphPad Software Inc., San Diego, CA, USA).ResultsThe anti-matriptase-2 monoclonal antibody KY1066 induces hepcidin expression and reduces iron overload in Hhb'n:<mice

[0130] To evaluate the activity of the anti-matriptase-2 antibody in reducing disease activity in p-thalassemic mice, 8-week-old Hbb&mice were treated with weekly IP injections of KYI 066 for 8 weeks and compared with Hbb^' treated with a non-relev ant antibody of the same isotype (control IgGl). Control Hbb^l+mice had elevated liver iron content compared to wild-ty pe mice at the same age, but similar, rather than lower, hepcidin levels (FIG. 2), indicating that, as previously reported (Oikonomidou and Rivella, 2018; Gardenghi et al., 2007), hepcidin can still respond partially to iron status in this model of P-thalassemia intermedia. Hbb&^+mice that received KY1066 had increased liver Hamp mRNA (x2.24 on average; 95% confidence interval: 1.51-3.33) and increased serum hepcidin levels compared to controls (FIG. 2, Panels A and B).KYI 066 was also effective in lowering serum iron levels (FIG. 2, Panel C) and bringing liver iron content to values in the normal range (FIG. 2. Panel D). Iron distribution in liver tissue sections from Hbb6'3^ and WT mice was assessed following Perl’s Prussian blue staining. Liver sections from Hbh'3'' mice treated with the control IgGl displayed dense blue punctate staining, representing abnormally high levels of insoluble iron stores termed hemosiderin. KYI 066 notably decreased liver iron staining when compared with control IgGl (FIG. 2, Panel E). These results provide evidence that, by neutralizing matriptase-2 activity, KYI 066 represents a potent in vivo modulator of Hamp expression, serum iron availability7, and liver iron accumulation in the mouse model of thalassemia intermedia. Similar effects were observed when KY1066 was given in combination with IP injections of RAP-536L, a modified activin receptor type IIB ligand trap, twice weekly, whereas, as expected from the different mode of action, treatment with RAP- 536L alone did not affect hepcidin mRNA (FIG. 2, Panel A), serum iron (FIG. 2, Panel C), or liver iron content (FIG. 2, Panels D and E). In the same line, despite increased erythropoietin (EPO) level, combination of KYI 066 and darbopoietin alpha treatmentsresulted anyway in increased hepcidin mRNA expression and reduced liver iron content (FIG. 3, Panels A and B)KY1066 ameliorates inejfective erythropoiesis in Hbbth3 / +mice

[0131] To assess whether KYI 066 ameliorated ineffective erythropoiesis in Hbb^'+mice, the different stages of ery throid differentiation in the spleen and bone marrow were identified by fluorescence activated cell sorting (FACS) analysis, using Teri 19 and CD44 cell surface markers. The additional parameter. FSC. was used to discriminate between five distinct erythroid populations: (I) pro-erythroblasts, (II) basophilic, (III) polychromatic, (IV) orthochromatic cells, and reticulocytes and (V) red blood cells. In the spleen, p-thalassemic erythroid cells exhibited increased cell proliferation (fractions II to IV) and reduced cell differentiation (fraction V) compared with those from normal mice (FIG. 4, Panel A) However, when the FACS profiles of control Hbb^l+mice and Hbb'3'' mice that received KYI 066 were compared, the latter group exhibited an improved profile, as indicated by a reduction in the number of cells in fractions III and IV, and an increase in terminally differentiated cells in fraction V (FIG. 4, Panel A). RAP-536L was slightly less effective, with more moderate reduction of cells in fraction IV and increase of red blood cells in fraction V. When administered together, KYI 066 and RAP-536L potentiated each other's effects, with more significant reduction of cells in fractions III and IV and increase in cells in fraction V (FIG. 4, Panel A). In the bone marrow, P-thalassemic erythroid cells exhibited increased cell numbers in fractions II and III and reduced terminally differentiated cells in fraction V (FIG. 4, Panel B). Both KY1066 and RAP-536L increased cells in fraction V. Most notably, the two treatments combined potentiated each other’s effects, with a distribution of erythroid precursors in the bone marrow of Hbb^ treated animals close to that seen in wild-type mice (FIG. 4, Panel B). Collectively, these results demonstrate that the neutralizing matriptase-2 antibody KYI 066 significantly enhances in vivo maturation and differentiation of erythroid cells in the spleen and, to a lesser extent, in the bone marrow of HbbiVil+mice. Interestingly, the combination of KYI 066 with RAP-536L entirely reverses ineffective erythropoiesis in the bone marrow.KY1066 limits the formation of toxic a-chain / heme aggregates and reduces ROS formation in Hbbth3,+mice

[0132] In (3-thalassemia, the unpaired a-globin chains precipitate with heme, forming large, insoluble aggregates in red blood cells, which increases ROS production and results in hemolysis. To test whether our treatments could reduce a-globin precipitates and thus improve the quality and survival of ery throid cells, membrane fractions were prepared from circulating red blood cells. The amount of membrane-bound globins in Hbr'f' control mice and Hbb&3!+mice that received the different treatments was assessed by' triton acetic urea (TAU) gel electrophoresis (FIG. 5, Panel A; FIG. 6). As expected, due to the unstable nature of free a-globins combined with relative (3-globin deficiency, a- globin precipitates were present in large amounts in (3-thalassemic erythrocytes.Compared with Hbf1'3control mice, those treated with RAP-536L exhibited a moderate reduction in membrane-associated a-globin aggregates. Interestingly, a-globin was undetectable in HbbiV3'^ mice treated with KYI 066 alone or in combination with RAP- 536L, as well as in wild-type membrane fractions. P-globin was undetectable in membrane fractions from all groups as expected. These results indicate that KYI 066 neutralizes toxic free a-globin precipitates even more effectively than RAP-536L. To determine whether the lower amount of a globin precipitates reduced ROS formation, a ROS indicator, 5-(and 6)-chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate, acyl ester (CM-H2DCFDA) was added to spleen and bone marrow cells. This compound permeates into the cells and is oxidized in the presence of ROS and free heme. Oxidation was detected by monitoring the increase in fluorescence by flow cytometry7. The analysis of the Teri 19+erythroid populations in the spleen and the bone marrow indicated that ROS were increased in Hbb'3'3mice compared with controls. Remarkably, mice treated with KYI 066 significantly reduced ROS to near normal levels in the spleen (FIG. 5, Panel B) but not in the bone marrow, in contrast to mice treated with RAP-536L (FIG. 5, Panel C). Further analysis using annexin V identified a large proportion of cells undergoing apoptosis in control Hbb'3'3mice compared with wild-type mice, both in the spleen and the bone marrow. KYI 066 significantly reduced the percentage of apoptotic (annexin V+) erythroid cells in the spleen (FIG. 5, Panel D) but not in the bone marrow (FIG. 5, Panel E), in contrast to RAP-536L that significantly reduced apoptosis in the bone marrow but not in the spleen. Collectively, these results demonstrate that theneutralizing matriptase-2 antibody KYI 066 reduces ROS formation and apoptosis in the spleen off / / ) / )1113mice. However, association with RAP-536L is needed to reach similar effects in the bone marrow.KY1066 improves reticulocytosis and splenomegaly in Hbbth3 / + mice

[0133] KYI 066 corrected reticulocytosis in Hbblh~ mice even more effectively than RAP-536L alone (FIG. 7, Panel A). Improvement of reticulocytosis was associated with a significant reduction in splenomegaly (FIG. 7, Panel B). Morphological inspection of the spleen indicated that decreased splenomegaly was associated with improved organ architecture, showing a normalization of the relative proportion of the white and red pulp (data not shown). However, and in contrast to RAP-536L, KYI 066 did not significantly increase hemoglobin levels, hematocrit, or red blood cell counts when compared to Hbb&^+controls (FIG. 7, Panels C-E). This is consistent with the fact that KYI 066 was less efficient in reducing apoptosis in the bone marrow. Notably, anemia end points were significantly improved when KY 1066 was given to Hbb&il+mice in association with RAP-536L (FIG. 7, Panels C-E) and in some extend with darbopoietin alpha (FIG. 7. Panels C-E).Discussion

[0134] Iron plays a major role in the deterioration of 0-thalassemia. Indeed, the high levels of transferrin saturation and iron delivered to erythroid progenitors are associated with production of hemi chromes that negatively affect erythropoiesis (Ginzburg & Rivella, 2011; Gardenghi et al., 2010; Li et al., 2010). As patients age. the size of the spleen increases and sequesters a growing number of damaged red blood cells. As a consequence, the number of erythroid progenitors expands in an attempt to compensate for the anemia, which negatively affects hepcidin expression and causes increased intestinal iron absorption and recycling despite replete iron stores. Increasing hepcidin expression can prevent or reduce iron overload, as well as break this vicious cycle (Parrow et al., 201 1). Suppression of Tmprss6 expression using anti-sense oligonucleotides (Guo et al., 2013; Casu et al., 2020; Casu et al., 2016b) or small interfering RNA (Schmidt et al., 2013; Vadolas et al., 2021; Schmidt et al.. 2015) in |3- thalassemic mice increased hepcidin and decreased iron loading. Interestingly, these treatments also improved erythropoiesis and anemia. Based on these findings, it has beensuggested that the use of these treatments could benefit thalassemic patients and several clinical trials are currently ongoing to assess their efficacy and safety (Motta et al.. 2020).

[0135] In this study, an alternative approach was considered to reach similar effects. This approach consists in blocking the protease activity of matriptase-2 via a monoclonal antibody binding to the serine protease active site. To obtain a fully human anti- matriptase 2 antibody, transgenic mice engineered to express the entire human immunoglobulin variable-gene repertoire (Lee et al., 2014) were used. These mice mount an effective immune response when challenged, producing high-affinity, fully human antibody. The use of fully human antibodies in patients avoids or reduces immunogenicity problems and thus is a favorable modality for long-term treatment of chronic diseases, which is particularly important for a disease such as (3-thalassemia. KYI 066 was identified through a multi-step screening cascade and blocks the protease activity of human and murine matriptase-2. When administered weekly to Hbb^^ mice, it was shown to induce hepcidin expression, reduce iron loading, prevent the formation of toxic a-chain / heme aggregates, reduce ROS formation, and improve reticulocytosis and splenomegaly. However, the ineffective erythropoiesis associated with this model and (3- thalassemia was not entirely corrected and, after 8 weeks of treatment, red blood cell counts as well as hemoglobin and hematocrit levels were still not improved.

[0136] To increase the effectiveness of KY1066 in (3-thalassemia treatment, a combinatorial approach was used. The matriptase-2 neutralizing antibody was administered in combination with RAP-536L, a ligand-trapping fusion protein known to sequestrate ligands of the TGF-p superfamily before they interact with their receptor (Suragani et al., 2014), thus inhibiting the Smad2 / 3 signal transduction cascade which plays an inhibitory role in terminal erythroid differentiation. The results indicate that RAP-536L treatment alone did not prevent iron overload but significantly reduced apoptosis in the erythroid populations of the bone marrow, normalized red blood cell counts, and improved hemoglobin and hematocrit levels. Interestingly, the association KY1066 with RAP-536L entirely reversed the (3-thalassemia phenotype inHbbth3 / +mice and corrected simultaneously iron overload, ineffective erythropoiesis, splenomegaly, and hematological parameters. Another interesting approach to increase the effectiveness of KYI 066 in (3-thalassemia treatment could also be to combine it with erythropoiesis enhancing agents like darbopoietin alpha to stimulate the production of RBC and improvethe anemia. Higher EPO level did not prevent the raise of hepcidin level induced by matriptase-2 inhibition and still prevented the iron overload. It is important to note that this combinatorial approach also improves anemia.

[0137] Thus, this study confirms that combinatorial treatments with agents eliciting different but complementary mechanisms of action are superior to single treatments. Moreover, this study shows that combination with luspatercept has a higher efficacy compared to combination with darbopoietin alpha.

[0138] KYI 066 is a first-in-class fully human monoclonal antibody that specifically neutralize matriptase-2. The very specific inhibitory' role of matriptase-2 on hepcidin production makes it, as shown in this study, a promising option for the management of secondary iron overload in (3-thalassemia, while minimizing the risk of any significant off-target effect and safety concerns. By restoring iron homeostasis in Hbbt'a‘i / +mice, this antibody markedly reduced levels of membrane-associated a-globin aggregates in erythrocytes, the main cause of ery throcytic damage and hemolysis in 0-thalassemia.This antibody could therefore also be helpful for the treatment of primary iron overload in diseases with insufficient hepcidin synthesis such as genetic hemochromatosis. Being fully human, it can be rapidly reformatted into a therapeutics that can be assessed in clinical trials for these important diseases. Because its association with RAP-536L ameliorates not only iron overload but also anemia, it might be an option in a short future to produce a multi-functional molecule consisting of the fusion of the KYI 066 IgG backbone structure with the ligand-trapping protein moiety containing the extracellular domain of activin receptor ty pe IIB, similar to Luspatercept (Cappellini et al., 2020). 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Claims

CLAIMS1. A method of treating one or more pathologies associated with P-thalassemia in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an effective amount of an anti-matriptase-2 antibody or antigen-binding fragment thereof.

2. A pharmaceutical composition comprising an effective amount of an anti- matriptase-2 antibody or antigen-binding fragment thereof for use in treating one or more pathologies associated with p-thalassemia in a subject in need thereof.

3. A method of alleviating one or more pathologies associated with P- thalassemia in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an effective amount of an anti-matriptase-2 antibody or antigen-binding fragment thereof.

4. A pharmaceutical composition comprising an effective amount of an anti- matriptase-2 antibody or antigen-binding fragment thereof for use in alleviating one or more pathologies associated with p-thalassemia in a subject in need thereof.

5. The method or pharmaceutical composition of any one of claims 1-4, wherein the one or more pathologies is selected from insoluble liver iron stores; a-globin precipitates in erythroid cells; reactive oxidized species (ROS) formation; apoptosis of erythroid cells; reticulocytosis: and a combination thereof.

6. The method or pharmaceutical composition of claim 5, wherein the one or more pathologies is a-globin precipitates in ery throcytes.

7. The method or pharmaceutical composition of claim 5, wherein the one or more pathologies is ROS formation in spleen, bone marrow, or a combination thereof.

8. The method or pharmaceutical composition of claim 5, wherein the one or more pathologies is apoptosis of erythroid cells in spleen, bone marrow, or a combination thereof.

9. The method or pharmaceutical composition of any one of claims 1-8, wherein the anti-matriptase-2 antibody or antigen-binding fragment thereof comprises(a) a heavy chain variable region comprising: a complementarity determining region (CDR)-l domain comprising an amino acid sequence of SEQ ID NO. 1, a CDR-2 domain comprising an amino acid sequence of SEQ ID NO. 2, and a CDR-3 domain comprising an amino acid sequence of SEQ ID NO. 3, and(b) a light chain variable region comprising: a CDR-1 domain comprising an amino acid sequence of SEQ ID NO. 4, a CDR-2 domain comprising an amino acid sequence of SEQ ID NO. 5, and a CDR-3 domain comprising an amino acid sequence of SEQ ID NO. 6.

10. The method or pharmaceutical composition of any one of claims 1-8, wherein the anti-matriptase-2 antibody or antigen-binding fragment thereof comprises(a) a heavy chain variable region comprising a complementarity determining region (CDR)-1 comprising an amino acid sequence of SEQ ID NO. 7, a CDR-2 domain comprising an amino acid sequence of SEQ ID NO. 8, and a CDR-3 domain comprising an amino acid sequence of SEQ ID NO. 9, and(b) a light chain variable region comprising a CDR-1 domain comprising an amino acid sequence of SEQ ID NO. 10, a CDR-2 domain comprising an amino acid sequence of SEQ ID NO. 11, and a CDR-3 domain comprising an amino acid sequence of SEQ ID NO. 6.

11. The method or pharmaceutical composition of any one of claims 1-10, wherein(a) the heavy chain variable region comprises an amino acid sequence at least 95% identical to SEQ ID NO. 12, and(b) the light chain variable region comprises an amino acid sequence at least 95% i dent cal to SEQ ID NO. 13.

12. The method or pharmaceutical composition of any one of claims 1-11, wherein(a) the heavy chain variable region comprises an amino acid sequence of SEQ ID NO.12, and(b) the light chain variable region comprises an amino acid sequence of SEQ ID NO.13.

13. The method or pharmaceutical composition of any one of claims 1-8, wherein the anti-matriptase-2 antibody or antigen-binding fragment thereof comprises:(a) heavy chain complementarity determining region (CDR)-l, CDR-2, and CDR-3 domains contained within a heavy chain variable region comprising an amino acid sequence of SEQ ID NO. 12, and(b) light chain CDR-1, CDR-2, and CDR-3 domains contained within a light chain variable region comprising an amino acid sequence of SEQ ID NO. 13.

14. The method or pharmaceutical composition of any one of claims 1-13, wherein(a) the heavy chain comprises an amino acid sequence of SEQ ID NO. 14, and(b) the light chain comprises an amino acid sequence of SEQ ID NO. 15.

15. The method or pharmaceutical composition of any one of claims 1-8, wherein the anti-matriptase-2 antibody or antigen-binding fragment thereof comprises(a) a heavy chain variable region comprising: a complementarity' determining region (CDR)-l domain comprising an amino acid sequence of SEQ ID NO. 1, a CDR-2 domain comprising an amino acid sequence of SEQ ID NO. 2, and a CDR-3 domain comprising an amino acid sequence of SEQ ID NO. 16, and(b) a light chain variable region comprising: a CDR-1 domain comprising an amino acid sequence of SEQ ID NO. 17, a CDR-2 domain comprising an amino acid sequence of SEQ ID NO. 5, and a CDR-3 domain comprising an amino acid sequence of SEQ ID NO. 18.

16. The method or pharmaceutical composition of any one of claims 1-8, wherein the anti-matriptase-2 antibody or antigen-binding fragment thereof comprises(a) a heavy chain variable region comprising a complementarity determining region (CDR)-1 comprising an amino acid sequence of SEQ ID NO. 19, a CDR-2 domain comprising an amino acid sequence of SEQ ID NO. 20, and a CDR-3 domain comprising an amino acid sequence of SEQ ID NO. 9, and(b) a light chain variable region comprising a CDR-1 domain comprising an amino acid sequence of SEQ ID NO. 21, a CDR-2 domain comprising an amino acid sequence of SEQ ID NO. 11, and a CDR-3 domain comprising an amino acid sequence of SEQ ID NO. 18.

17. The method or pharmaceutical composition of any one of claims 1-8, 15, or 16, wherein(a) the heavy chain variable region comprises an amino acid sequence at least 95% identical to SEQ ID NO. 22, and(b) the light chain variable region comprises an amino acid sequence at least 95% identical to SEQ ID NO. 23.

18. The method or pharmaceutical composition of any one of claims 1-8 or 15-17, wherein(a) the heavy chain variable region comprises an amino acid sequence of SEQ ID NO.22, and(b) the light chain variable region comprises an amino acid sequence of SEQ ID NO.23.

19. The method or pharmaceutical composition of any one of claims 1-8, wherein the anti-matriptase-2 antibody or antigen-binding fragment thereof comprises:(a) heavy chain complementarity determining region (CDR)-l, CDR-2, and CDR-3 domains contained within a heavy chain variable region comprising an amino acid sequence of SEQ ID NO. 22, and(b) light chain CDR-1, CDR-2, and CDR-3 domains contained within a light chain variable region comprising an amino acid sequence of SEQ ID NO. 23.

20. The method or pharmaceutical composition of any one of claims 1-8 or 15-19, wherein(a) the heavy chain comprises an amino acid sequence of SEQ ID NO. 24, and(b) the light chain comprises an amino acid sequence of SEQ ID NO. 25.

21. The method or pharmaceutical composition of any one of claims 1-8, wherein the anti-matriptase-2 antibody or antigen-binding fragment thereof comprises(a) a heavy chain variable region comprising: a complementarity determining region (CDR)-l domain comprising an amino acid sequence of SEQ ID NO. 26, a CDR-2 domain comprising an amino acid sequence of SEQ ID NO.

27. and a CDR-3 domain comprising an amino acid sequence of SEQ ID NO. 28, and(b) a light chain variable region comprising: a CDR-1 domain comprising an amino acid sequence of SEQ ID NO. 29, a CDR-2 domain comprising an amino acid sequence of SEQ ID NO. 5, and a CDR-3 domain comprising an amino acid sequence of SEQ ID NO. 30.

22. The method or pharmaceutical composition of any one of claims 1-8, wherein the anti-matriptase-2 antibody or antigen-binding fragment thereof comprises(a) a heavy chain variable region comprising a complementarity determining region (CDR)-l comprising an amino acid sequence of SEQ ID NO.

31. a CDR-2 domain comprising an amino acid sequence of SEQ ID NO. 32, and a CDR-3 domain comprising an amino acid sequence of SEQ ID NO. 33, and(b) a light chain variable region comprising a CDR-1 domain comprising an amino acid sequence of SEQ ID NO. 34, a CDR-2 domain comprising an amino acid sequence of SEQ ID NO. 35, and a CDR-3 domain comprising an amino acid sequence of SEQ ID NO. 30.

23. The method or pharmaceutical composition of any one of claims 1-8, 21, or 22. wherein(a) the heavy chain variable region comprises an amino acid sequence at least 95% identical to SEQ ID NO. 36, and(b) the light chain variable region comprises an amino acid sequence at least 95% identical to SEQ ID NO. 37.

24. The method or pharmaceutical composition of any one of claims 1-8 or 21-23, wherein(a) the heavy chain variable region comprises an amino acid sequence of SEQ ID NO.

36. and(b) the light chain variable region comprises an amino acid sequence of SEQ ID NO.37.

25. The method or pharmaceutical composition of any one of claims 1-8, wherein the anti-matriptase-2 antibody or antigen-binding fragment thereof comprises:(a) heavy chain complementarity determining region (CDR)-l, CDR-2, and CDR-3 domains contained within a heavy chain variable region comprising an amino acid sequence of SEQ ID NO. 36, and(b) light chain CDR-1, CDR-2, and CDR-3 domains contained within a light chain variable region comprising an amino acid sequence of SEQ ID NO. 37.

26. The method or pharmaceutical composition of any one of claims 1-8 or 21-25, wherein(a) the heavy chain comprises an amino acid sequence of SEQ ID NO. 38, and(b) the light chain comprises an amino acid sequence of SEQ ID NO. 39.

27. The method or pharmaceutical composition of any one of claims 1-8, wherein the anti-matriptase-2 antibody or antigen-binding fragment thereof comprises(a) a heavy chain variable region comprising: a complementarity determining region (CDR)-l domain comprising an amino acid sequence of SEQ ID NO. 40, a CDR-2 domain comprising an amino acid sequence of SEQ ID NO. 41, and a CDR-3 domain comprising an amino acid sequence of SEQ ID NO. 42, and(b) a light chain variable region comprising: a CDR-1 domain comprising an amino acid sequence of SEQ ID NO. 43, a CDR-2 domain comprising an amino acid sequence ofSEQ ID NO. 44, and a CDR-3 domain comprising an amino acid sequence of SEQ ID NO. 45.

28. The method or pharmaceutical composition of any one of claims 1-8, wherein the anti-matriptase-2 antibody or antigen-binding fragment thereof comprises(a) a heavy chain variable region comprising a complementarity determining region (CDR)-l comprising an ammo acid sequence of SEQ ID NO.

46. a CDR-2 domain comprising an amino acid sequence of SEQ ID NO. 47, and a CDR-3 domain comprising an amino acid sequence of SEQ ID NO. 48, and(b) a light chain variable region comprising a CDR-1 domain comprising an amino acid sequence of SEQ ID NO. 49, a CDR-2 domain comprising an amino acid sequence of SEQ ID NO. 50, and a CDR-3 domain comprising an amino acid sequence of SEQ ID NO. 45.

29. The method or pharmaceutical composition of any one of claims 1-8, 27, or 28, wherein(a) the heavy chain variable region comprises an amino acid sequence at least 95% identical to SEQ ID NO. 51, and(b) the light chain variable region comprises an amino acid sequence at least 95% identical to SEQ ID NO. 52.

30. The method or pharmaceutical composition of any one of claims 1-8 or 27-29, wherein(a) the heavy chain variable region comprises an amino acid sequence of SEQ ID NO.51, and(b) the light chain variable region comprises an amino acid sequence of SEQ ID NO.52.

31. The method or pharmaceutical composition of any one of claims 1-8, wherein the anti-matriptase-2 antibody or antigen-binding fragment thereof comprises:(a) heavy chain complementarity determining region (CDR)-l, CDR-2, and CDR-3 domains contained within a heavy chain variable region comprising an amino acid sequence of SEQ ID NO. 51, and(b) light chain CDR-1, CDR-2, and CDR-3 domains contained within a light chain variable region comprising an amino acid sequence of SEQ ID NO. 52.

32. The method or pharmaceutical composition of any one of claims 1-8 or 27-31, wherein(a) the heavy chain comprises an amino acid sequence of SEQ ID NO. 53, and(b) the light chain comprises an amino acid sequence of SEQ ID NO. 54.

33. The method or pharmaceutical composition of any one of claims 1-32, wherein the method further comprises administering, or the use is in combination with, an antagonist of a transforming growth factor-beta (TGF-P) superfamily ligand.

34. The method or pharmaceutical composition of claim 33, wherein the antagonist is an activin II receptor ligand trap.

35. . The method or pharmaceutical composition of claim 34, wherein the antagonist is an activin MB receptor Fc fusion protein.

36. The method or pharmaceutical composition of claim 35, wherein the antagonist is luspatercept.

37. The method or pharmaceutical composition of claim 34, wherein the antagonist is an activin II A receptor Fc fusion protein38. The method or pharmaceutical composition of claim 37. wherein the antagonist is sotatercept.

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