An expression system for producing recombinant haptoglobin (Hp) beta chain

Abstract

The present invention relates to an expression system for producing recombinant haptoglobin (Hp) beta chain or a hemoglobin-binding fragment thereof, a recombinant Hp molecule, and its use for treating and / or preventing conditions associated with cell-free hemoglobin (Hb).

Description

[Technical field]

[0001] The present invention relates generally to an expression system for producing recombinant haptoglobin (Hp) beta chain or hemoglobin-binding fragments thereof, recombinant Hp molecules, and their use for treating and / or preventing conditions associated with abnormalities in cell-free hemoglobin (Hb) levels. [Background technology]

[0002] Erythrocyte lysis is characterized by the rupture of red blood cells (erythocytes) resulting in the release of hemoglobin (Hb) into the plasma and is a common cause of enzyme deficiencies, hemoglobinopathies (e.g., thalassemia), hereditary spherocytosis, paroxysmal nocturnal hemoglobinuria, and spur cell lysis. In addition to red blood cell abnormalities such as hemolytic anemia, splenomegaly, autoimmune disorders (e.g., hemolytic disease of the newborn), genetic disorders (e.g., sickle cell disease or G6PD deficiency), microangiopathic hemolysis, gram-positive bacterial infections (e.g., Streptococcus, Enterococcus, and Staphylococcus), parasitic infections (e.g., Plasmodium), toxins, trauma (e.g., burns), hemorrhagic stroke, sepsis, atherosclerosis, transfusions (especially massive transfusions), and anemic disorders are prominent features of anemic disorders in patients using cardiopulmonary bypass (see, e.g., J. Med. Soc. 2002; 2003; 2004; and 2005).

[0003] The adverse effects seen in patients with conditions associated with hemolysis are largely attributed to the release of iron and iron-containing compounds from red blood cells, such as Hb and heme. Under physiological conditions, acellular hemoglobin is typically bound to soluble proteins such as haptoglobin (Hp) (see Non-Patent Document 6) and transported to macrophages and hepatocytes. However, under circumstances in which the occurrence of hemolysis accelerates and / or takes on a pathological character, the buffering capacity of Hp is overwhelmed. As a result, Hb is rapidly oxidized to ferric hemoglobin, which releases free heme (containing protoporphyrin IX and iron; see Non-Patent Document 7). Although heme plays a vital role in several biological processes (e.g., as part of essential proteins such as hemoglobin and myoglobin), free heme is highly toxic. For example, free heme is a source of redox-active iron, which produces highly toxic reactive oxygen species (ROS) that damage lipid membranes (see Non-Patent Document 8), proteins, and nucleic acids. Heme toxicity is further exacerbated by its ability to intervene in lipid membranes, where it causes oxidation of membrane components and promotes cell lysis and death (see Non-Patent Document 9).

[0004] Evolutionary pressure from exposure to sustained low levels of extracellular Hb / heme has led to compensatory mechanisms that control the deleterious effects of free Hb / heme under physiological steady state and during mild hemolysis. These systems include the release of a group of plasma proteins that bind Hb or heme, including the Hb scavenger Hp, and heme scavenger proteins such as hemopexin (Hpx) and α1-microglobin (10).

[0005] As mentioned above, plasma Hp acts as a scavenger for cell-free Hb, binding to cell-free Hb to form a neutralizing Hb:Hp complex (see Non-Patent Document 11). However, when the amount of Hb exceeds the scavenging capacity of plasma Hp, the local accumulation of Hb, especially in blood vessels and renal tissues, results in oxidative stress that may lead to adverse secondary outcomes for patients. The protection provided by Hp attenuates at least two toxicological consequences of Hb. First, the large molecular size of the Hb:Hp complex prevents extravasation of cell-free Hb. This mechanism protects renal function and preserves intravascular nitric oxide (NO) homeostasis by limiting the access of free Hb to the vascular wall (see Non-Patent Document 12). Second, the formation of the Hb:Hp complex stabilizes the structure of the Hb molecule in a way that limits the transfer of heme from its globin chains to proteins and reactive lipids (see Non-Patent Document 7). These mechanisms largely contribute to the antioxidant function of Hp following hemolysis.

[0006] Endogenous Hp may provide significant protection against cell-free Hb toxicity, but is rapidly consumed and depleted during more pronounced acute or prolonged hemolysis (see Non-Patent Document 13). Therefore, Hp replacement is considered a therapeutic modality that supports preclinical proof-of-concept in vitro and in animal models of hemolysis. Preclinical studies have mostly assessed the therapeutic potential of Hp purified from pooled human plasma fractions. However, this approach has several limitations that are relevant to clinical practice, such as (1) a mixture of different Hp phenotypes (1-1, 2-1, and 2-2) may induce neutralizing antibody responses in some patients with prolonged replacement therapy; (2) different phenotypes may have different efficacies; and (3) phenotypic forms may support different pharmacokinetics. Therefore, considering the potential limitations of plasma-derived Hp, recombinant protein production may provide a reasonable therapeutic strategy that avoids or otherwise alleviates at least some of the aforementioned limitations of plasma-derived Hp. In addition, recombinant protein production strategies may provide therapeutic agents with enhanced functionality, bioavailability, and pharmacokinetics. However, recent attempts to produce recombinant Hp by expressing precursor molecules (proHp) have been noted to reduce binding to Hb (see Non-Patent Document 14). Thus, there remains a need for alternative or improved therapies to treat and / or prevent conditions associated with cell-free Hb, in which the Hb scavenging properties of Hp would be beneficial. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Hoppe et al. (1998, Curr Opin Pediatr, 10(1):49~52) [Non-Patent Document 2] Roumenina (2016, Trends in Molecular Medicine, 22(3):200~213) [Non-Patent Document 3] Merle (2019, PNAS, 116(13):6280~6285) [Non-Patent Document 4] Larsen (2010, Science Translational Medicine:2(51):51~71) [Non-Patent Document 5] Balla G (2019, Int J Mol Sci, 20(15):3675) [Non-Patent Document 6] CBFAndersen et al., 2012, Nature, 489(7416):456-459 [Non-Patent Document 7] Schaer et al. (2014, Frontiers in PHYSIOLOGY, 5:1~13) [Non-Patent Document 8] Deuel et al. (2015, Free Radical Biology and Medicine, 89:931~943 [Non-Patent Document 9] Jeney et al. (2002, Blood, 100(3):879-87) [Non-Patent Document 10] Schaer et al., 2013, Blood, 121(8):1276-84 [Non-Patent Document 11] Shim et al., 1965, Nature, 207:1264-1267 [Non-Patent Document 12] Azarov et al., 2008, Nitric Oxide, 18(4):296~302 [Non-Patent Document 13] Boretti et al., 2014, Frontiers in Physiology, 5:385 [Non-Patent Document 14] Heinderyckx et al., 1988~1989, Mol Biol Rep, 13(4):225~32 Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention is based, at least in part, on the surprising discovery by the inventors that functional haptoglobin beta chain or hemoglobin-binding fragments thereof can be generated from N-terminally truncated prohaptoglobin (proHp) in a mammalian expression system. Furthermore, the N-terminally truncated proHp can be advantageously modified to carry functional moieties such as Hpx, Fc, or albumin, thereby generating constructs with improved therapeutic properties. [Means for solving the problem]

[0009] Thus, in one aspect disclosed herein, there is provided an expression system for producing recombinant haptoglobin beta chain or a hemoglobin-binding fragment thereof in a mammalian cell, comprising: (a) a first nucleic acid sequence encoding an N-terminally truncated prohaptoglobin (proHp), the N-terminally truncated proHp comprising (i) at least 14 consecutive C-terminal amino acid residues of a haptoglobin alpha chain, and (ii) a haptoglobin beta chain, or a hemoglobin-binding fragment thereof, the N-terminally truncated proHp comprising an internal enzymatic cleavage site between the at least 14 consecutive C-terminal amino acid residues of the haptoglobin alpha chain and the haptoglobin beta chain, or a hemoglobin-binding fragment thereof; and (b) a second nucleic acid sequence encoding an enzyme capable of cleaving the N-terminal truncated proHp at the enzyme cleavage site; Includes; When the first and second nucleic acid sequences are introduced into a mammalian cell and the N-terminal truncated proHp and the enzyme are then expressed in the cell, the enzyme is capable of cleaving the N-terminal truncated proHp at the internal enzyme cleavage site, thereby releasing the haptoglobin beta chain or a hemoglobin-binding fragment thereof from the N-terminal truncated proHp. An expression system is provided.

[0010] In another aspect disclosed herein, there is provided an expression vector for producing a recombinant haptoglobin beta chain or a hemoglobin-binding fragment thereof in a mammalian cell, comprising: (a) a first nucleic acid sequence, as described herein; and (b) a second nucleic acid sequence, as described herein. An expression vector comprising the

[0011] The present disclosure also extends to mammalian cells comprising the expression systems or expression vectors described herein.

[0012] In another aspect disclosed herein, there is provided a method of making a recombinant haptoglobin beta chain or a hemoglobin binding fragment thereof, comprising: (a) introducing into a mammalian cell an expression system as described herein to produce a modified mammalian cell; (b) culturing the modified mammalian cells produced in step (a) under conditions and for a period of time sufficient to allow for the production of a recombinant haptoglobin beta chain or a hemoglobin-binding fragment thereof; and (c) recovering the recombinant haptoglobin beta chain or hemoglobin-binding fragment thereof produced in step (b). A method is provided that includes:

[0013] In another aspect disclosed herein, there is provided a recombinant haptoglobin beta chain, or a hemoglobin-binding fragment thereof, produced by the methods described herein.

[0014] In another aspect disclosed herein, a recombinant hemoglobin-binding molecule is provided that comprises: (i) a haptoglobin beta chain, or a hemoglobin-binding fragment thereof; and (ii) an N-terminally truncated haptoglobin alpha chain, wherein the N-terminally truncated haptoglobin alpha chain comprises at least 14 contiguous C-terminal amino acid residues of the haptoglobin alpha chain, and wherein the at least 14 contiguous C-terminal amino acid residues of the haptoglobin alpha chain are non-contiguous with the haptoglobin beta chain, or a hemoglobin-binding fragment thereof, and wherein the N-terminally truncated haptoglobin alpha chain is joined to the haptoglobin beta chain, or a hemoglobin-binding fragment thereof.

[0015] The present disclosure also extends to a pharmaceutical composition comprising a therapeutically effective amount of a recombinant hemoglobin binding molecule described herein, or a recombinant haptoglobin beta chain or hemoglobin binding fragment thereof described herein, and a pharma- ceutically acceptable carrier.

[0016] In another aspect disclosed herein, a method is provided for treating or preventing a condition associated with acellular hemoglobin (Hb) in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a recombinant hemoglobin binding molecule as described herein, or a recombinant haptoglobin beta chain or hemoglobin binding fragment thereof as described herein, for a period of time sufficient to allow the haptoglobin beta chain or hemoglobin binding fragment thereof to form a complex with the acellular Hb, thereby neutralizing the acellular Hb. In an embodiment, the condition is associated with red blood cell lysis.

[0017] Also disclosed herein is a pharmaceutical composition for use in treating or preventing a condition associated with acellular hemoglobin (Hb) in a subject, comprising a therapeutically effective amount of a recombinant hemoglobin binding molecule described herein, or a recombinant haptoglobin beta chain or hemoglobin binding fragment thereof described herein, and a pharma- ceutical acceptable carrier.

[0018] In another aspect disclosed herein, there is provided the use of a therapeutically effective amount of a recombinant hemoglobin binding molecule described herein, or a recombinant haptoglobin beta chain or hemoglobin binding fragment thereof described herein, in the manufacture of a medicament for the treatment or prevention of a condition associated with acellular hemoglobin (Hb) in a subject.

[0019] The present disclosure also extends to a therapeutically effective amount of a recombinant hemoglobin-binding molecule as described herein, or a recombinant haptoglobin beta chain or hemoglobin-binding fragment thereof as described herein, for use in treating or preventing a condition associated with acellular hemoglobin (Hb) in a subject.

[0020] All references cited herein, including any patents or patent applications, are incorporated herein by reference to enable a full understanding of the present invention. However, the reference herein to any prior publication (or information derived therefrom) or any known subject matter is not, and should not be understood as, an admission or acceptance, or any form of suggestion, that this prior publication (or information derived therefrom) or known subject matter forms part of the common general knowledge in the field of the subject matter to which this specification relates.

[0021] Embodiments of the present invention will now be described with reference to the following drawings, which are intended to be exemplary only. [Brief description of the drawings]

[0022] [Figure 1] FIG. 1 shows the structure of an illustrative example of human haptoglobin; (A) Schematic representation of human Hp1 and human Hp2. Amino acid sequences common to the α-chains of Hp1 and Hp2 are shown and highlighted in green (SEQ ID NOs: 14 and 17). The amino acid sequence shaded in blue within the α-chain of Hp2 (second line [middle] of the Hp2 sequence) determines a strikingly different molecular phenotype. The asterisk designates the cysteine ​​residue required for disulfide bond formation. The arrows indicate the C1rLP cleavage site. (B) Protein quaternary structures of the Hp1-1 homodimer with one inter-α-chain disulfide bond and three mutant Hp2-2 cyclic homomultimers with two inter-α-chain disulfide bonds. [Diagram 2] FIG. 1 depicts the spectral deconvolution to follow the transition of heme-albumin to heme-Hpx. A series of UV-VIS spectra was recorded over time (3 hours at 37° C.) for a reaction mixture containing 12.5 μM heme-albumin and human Hpx. The first spectrum (t=0) is highlighted in orange (light) and the last spectrum (after t=3 hours) is highlighted in blue (dark). [Figure 3-1] (A) Amino acid sequence of human prohaptoglobin 2FS. The signal peptide (amino acid residues 1-18; MSALGAVIALLLWGQLFA; SEQ ID NO: 15) is highlighted in yellow. The C1rLP cleavage site after Arg161(R) is indicated by an arrow. The alpha (α) chain is highlighted in blue (amino acid residues 19-161) and the beta (β) chain is highlighted in light green (amino acid residues 162-406). Cysteine ​​residues that form inter- and intrachain disulfide bonds are at amino acid positions 33, 52, 86, 92, 149, 266, 309, 340, 351, and 381. The CD163 binding site is identified by amino acid residues 318, 320, 322, and 323. The amino acid residues of the variants described herein are numbered with +1 as the initiating methionine and are based on the amino acid sequence of Hp2FS. In this regard, amino acids Asp70, Lys71, Asn129, and Glu130 are highlighted. (B) Schematic representation of the processing of the prohaptoglobin 2FS polypeptide chain into alpha and beta chains. The positions of the inter- and intrachain disulfide bonds (SS) are indicated. (C) Coomassie stained reducing SDS-PAGE of rHp1S and rHp2FS produced in transfected FS293F cells. The Hpα chain appears at 12 or 19 kDa, and the Hpβ chain at 47 kDa. In addition, uncleaved proHp1 and proHp2 appear at their predicted sizes of 53 or 57 kDa. Coexpression of C1r-LP(+) efficiently cleaves all proHp into its subunits. C1r-LP appears at 68 kDa. (D) Anti-8His Western blot showing uncleaved proHp in the absence of C1r-LP, and the small Hp β chain, as well as His-tagged protease in the presence of coexpression of C1r-LP. [Figure 3-2] Continued from Figure 3-1. [Figure 3-3] Continued from Figure 3-2. [Diagram 3-4] Continued from Figure 3-3. [Figure 4-1]Expression and purification of haptoglobin beta fragments in Expi293F cells. (A) Schematic depicting recombinant beta fragment constructs, each with a C-terminal 8xHis tag. The amino acids and intrachain disulfide bond (SS) positions are indicated. (B) Schematic depicting recombinant beta fragment constructs with a C-terminal 8xHis tag and an additional 14 amino acids. Processing of the preprotein by C1r-LP, amino acids, and the interchain and intrachain disulfide bond (SS) positions are indicated. (C) (Left panel) Coomassie stained reducing SDS-PAGE of recombinant Hp beta fragment constructs made in transiently transfected Expi293F cells. Co-transfection of constructs encoding the N-terminally extended beta-fragment Hu haptoglobin 2FS(148-406)-8His with constructs encoding Hu-C1r-LP-FLAG was performed to confirm cleavage of the nascent polypeptide. (Middle panel) Anti-His Western blot versus reducing SDS-PAGE of recombinant Hp beta-fragment constructs made in transiently transfected Expi293F cells. (Right panel) Anti-Hp Western blot versus reducing SDS-PAGE of recombinant Hp beta-fragment constructs made in transiently transfected Expi293F cells. (D) (Left panel) Analytical SEC chromatogram of nickel affinity purified N-terminal extended Huhaptoglobin 2FS(148–406)-8His performed on an Agilent 1260 Infinity HPLC with a Superdex 200 Increase 5 / 150 column and MT-PBS mobile phase. (Right panel) 4–12% Bis-Tris SDS-PAGE gel showing the characteristic glycoform of haptoglobin, a doublet band, migrating above its background molecular weight of 29.9 kDa. [Figure 4-2] Continued from Figure 4-1. [Figure 4-3] Continued from Figure 4-2. [Figure 4-4] Continued from Figure 4-3. [Diagram 5] Figure 1 presents a schematic showing the molecular design of haptoglobin beta fragment fusion proteins. (A) Schematic depicting recombinant Hp beta fragment constructs (amino acids 162-406) with either an N-terminal or C-terminal fusion partner. The amino acids and the position of the intrachain disulfide bonds (SS) are indicated. (B) Schematic depicting recombinant Hp beta fragment constructs (amino acids 148-406) with either an N-terminal or C-terminal fusion partner with an additional N-terminal 14 amino acids. Processing of the preprotein by C1r-LP. The amino acids and the position of the interchain and intrachain disulfide bonds (SS) are indicated. [Figure 6-1]Figure 2: Expression and purification of Hu hemopexin-Hu haptoglobin beta fusion protein in Expi293F cells. (A) Schematic diagram depicting recombinant beta fragment constructs fused to (i) human hemopexin (Hpx; amino acids 1-462) at the N-terminus followed by a Gly-Ser linker and then amino acids 162-406; (ii) human Hp beta fragment encoding amino acids 162-406, in which the unpaired cysteine ​​at amino acid 266 has been mutated to alanine; (iii) human Hp beta fragment encoding amino acids 148-406, retaining the C1r-LP cleavage site and the cysteine ​​required for intrachain disulfide bonds. The positions of the amino acids and interchain disulfide bonds (SS) are indicated. (B) (Left panel) Coomassie stained reduced SDS-PAGE of recombinant Hpx-Hp beta fragment constructs made in transiently transfected Expi293F cells. Co-transfection with constructs encoding the N-terminally extended beta fragments Hu haptoglobin 2FS(148-406) and Hu-C1r-LP-FLAG was performed to confirm cleavage of the nascent polypeptide. (Middle panel) Anti-His Western blot on reduced SDS-PAGE of recombinant Hpx-Hp beta fragment constructs made in transiently transfected Expi293F cells. (Right panel) Anti-Hp Western blot on reduced SDS-PAGE of recombinant Hp beta fragment constructs made in transiently transfected Expi293F cells. (C) Analysis of aggregate content and protein processing by SEC and SDS-PAGE. (i) Preparative SEC chromatogram of nickel affinity purified HuHemopexin-HuHaptoglobin2FS(162-406)-8His performed on a Superdex 200 16 / 600 column and an AKTAxpress system with MT-PBS mobile phase. The arrow indicates the peak containing the fusion protein of the predicted size.(ii) Analytical SEC chromatogram of nickel affinity purified N-terminal extended Hu hemopexin-Hu haptoglobin 2FS(148-406)-8His / Hu-C1r-LP-FLAG performed on an Agilent 1260 Infinity HPLC system with a Superdex 200 Increase 5 / 150 column and MT-PBS mobile phase. The arrow indicates the peak containing the fusion protein of the predicted size. (iii) Reducing and non-reducing SDS-PAGE gels (4-12% Bis-Tris) showing the purity and proper processing of nickel affinity purified Hu hemopexin-Hu haptoglobin 2FS(148-406)-8His. [Figure 6-2] Continued from Figure 6-1. [Figure 6-3] Continued from Figure 6-2. [Figure 6-4] Continued from Figure 6-3. [Figure 7-1]Expression and purification of HSA-Hu haptoglobin beta fusion protein in Expi293F cells. (A) Schematic depicting recombinant beta fragment constructs containing human serum albumin (HSA) at the N-terminus and fused to (i) a human Hp beta fragment encoding amino acids 162-406 followed by a Gly-Ser linker; (ii) a human Hp beta fragment encoding amino acids 162-406 followed by a Gly-Ser linker, with the unpaired cysteine ​​at amino acid 266 mutated to alanine; (iii) a human Hp beta fragment encoding amino acids 148-406, retaining the C1r-LP cleavage site and the cysteine ​​required for the intrachain disulfide bond. The positions of the amino acids and interchain disulfide bonds (SS) are indicated. (B) (Left panel) Coomassie stained reduced SDS-PAGE of recombinant HSA-Hp beta fragment constructs made in transiently transfected Expi293F cells. Co-transfection of constructs encoding the N-terminally extended beta-fragment Hu haptoglobin 2FS(148-406) and Hu-C1r-LP-FLAG was performed to confirm cleavage of the nascent polypeptide. (Middle panel) Anti-HSA Western blot versus reducing SDS-PAGE of recombinant HSA-Hp beta-fragment constructs made in transiently transfected Expi293F cells. (Right panel) Anti-Hp Western blot versus reducing SDS-PAGE of recombinant Hp beta-fragment constructs made in transiently transfected Expi293F cells. (C) Analysis of aggregate content and protein processing by SEC and SDS-PAGE. (i) Preparative SEC chromatogram of HSA affinity purified HSA-GS13-Hu haptoglobin (162-406) performed on a Superdex 200 16 / 600 column and an AKTAxpress system with MT-PBS mobile phase. The arrow indicates the peak containing the fusion protein of the predicted size.(ii) Analytical SEC chromatogram of HSA affinity purified N-terminal extended HSA-HuHaptoglobin2FS(148-406) / Hu-C1r-LP-FLAG performed on an Agilent 1260 Infinity HPLC system with a Superdex 200 Increase 5 / 150 column and MT-PBS mobile phase. The arrow indicates the peak containing the fusion protein of the predicted size. (iii) Reducing and non-reducing SDS-PAGE gels (4-12% Bis-Tris) showing the purity and proper processing of HSA affinity purified HSA-HuHaptoglobin2FS(148-406). [Figure 7-2] Continued from Figure 7-1. [Figure 7-3] Continued from Figure 7-2. [Figure 7-4] Continued from Figure 7-3. [Figure 7-5] Continued from Figure 7-4. [Figure 8-1]Expression and purification of Fc-Hu haptoglobin beta fusion protein in Expi293F cells. (A) Schematic depicting recombinant beta fragment constructs containing (i) human IgG1Fc fused to the N-terminus of human Hp beta fragment encoding amino acids 162-406; (ii) mouse IgG2a followed by human Hp beta fragment encoding amino acids 148-406, retaining the C1r-LP cleavage site and the cysteine ​​required for intrachain disulfide bond. Positions of amino acids and interchain disulfide bonds (SS) are indicated. (B) (left panel) Coomassie stained reducing SDS-PAGE of recombinant Fc-Hp beta fragment constructs made in transiently transfected Expi293F cells. Co-transfection of constructs encoding the N-terminally extended beta-fragment Hu haptoglobin 2FS(148-406) and Hu-C1r-LP-FLAG was performed to confirm cleavage of the nascent polypeptide. (Middle panel) Anti-Fc Western blot versus reducing SDS-PAGE of recombinant Fc-Hp beta-fragment constructs made in transiently transfected Expi293F cells. (Right panel) Anti-Hp Western blot versus reducing SDS-PAGE of recombinant Hp beta-fragment constructs made in transiently transfected Expi293F cells. (C) Analysis of aggregate content and protein processing by SEC and SDS-PAGE. (i) Analytical SEC chromatogram of Protein A affinity purified N-terminal extended muIgG2aFc-Hu haptoglobin 2FS(148-406) / Hu-C1r-LP-FLAG performed on an Agilent 1260 Infinity system with a Superdex 200 Increase 5 / 150 column and MT-PBS mobile phase. The arrow indicates the peak containing the fusion protein of the predicted size.(ii) Reducing and non-reducing SDS-PAGE gels (4-12% Bis-Tris) showing the purity and correct processing of Protein A affinity purified, muIgG2aFc-Hu haptoglobin 2FS(148-406). [Figure 8-2] Continued from Figure 8-1. [Figure 8-3] Continued from Figure 8-2. [Figure 8-4] Continued from Figure 8-3. [Figure 9-1]Expression and purification of hemopexin-MSA-Hu haptoglobin beta fusion protein in Expi293F cells. (A) Schematic depicting a recombinant Hp beta fragment construct containing human hemopexin (Hpx; amino acids 1-462) at the N-terminus followed by mouse serum albumin (msa) and then fused to i) a human Hp beta fragment encoding amino acids 162-406; ii) a human Hp beta fragment encoding amino acids 148-406, retaining the C1r-LP cleavage site and the cysteine ​​required for intrachain disulfide bonds. The positions of the amino acids and interchain disulfide bonds (SS) are indicated. (B) (Left panel) Coomassie stained reduced SDS-PAGE of the recombinant Hpx-msa-Hp beta fragment construct made in transiently transfected Expi293F cells. Co-transfection of constructs encoding the N-terminally extended beta-fragment Hu haptoglobin 2FS(148-406) and Hu-C1r-LP-FLAG was performed to confirm cleavage of the nascent polypeptide. (Middle panel) Anti-MSA Western blot versus reducing SDS-PAGE of recombinant Fc-Hp beta-fragment constructs made in transiently transfected Expi293F cells. (Right panel) Anti-Hp Western blot versus reducing SDS-PAGE of recombinant Hp beta-fragment constructs made in transiently transfected Expi293F cells. (C) Analysis of aggregate content and protein processing by SEC and SDS-PAGE. (i) Preparative SEC chromatogram of CaptureSelect HSA affinity purified HuHemopexin-HSA-HuHaptoglobin2FS(162-406) performed on a Superdex 200 16 / 600 column and AKTAxpress system with MT-PBS mobile phase. The arrow indicates the peak containing the fusion protein of the predicted size.(ii) Analytical SEC chromatogram of Mimetic Blue affinity purified N-terminal extended Hu hemopexin-MSA-Hu haptoglobin 2FS(148-406) / Hu-C1r-LP-FLAG performed on an Agilent 1260 Infinity HPLC system with a Superdex 200 Increase 5 / 150 column and MT-PBS mobile phase. The arrow indicates the peak containing the fusion protein of the predicted size. (iii) Reducing and non-reducing SDS-PAGE gels (4-12% Bis-Tris) showing the purity and proper processing of Mimetic Blue affinity purified Hu hemopexin-MSA-Hu haptoglobin 2FS(148-406). [Figure 9-2] Continued from Figure 9-1. [Figure 9-3] Continued from Figure 9-2. [Figure 9-4] Continued from Figure 9-3. [Figure 10-1]Expression and purification of Hu hemopexin-mIgG2aFc-Hu haptoglobin 2FS(148-406) fusion protein in Expi293F cells. (A) Schematic depicting recombinant Hp beta fragment construct containing human hemopexin (Hpx; amino acids 1-462) at the N-terminus followed by a Gly-Ser linker, mouse IgG2aFc, then fused to human Hp beta fragment encoding amino acids 148-406, bearing the C1r-LP cleavage site and the cysteine ​​required for intrachain disulfide bonds. The positions of amino acids and interchain disulfide bonds (SS) are indicated. (B) (Left panel) Coomassie stained reduced SDS-PAGE of recombinant Hu hemopexin-mIgG2aFc-Hu haptoglobin 2FS(148-406) construct made in transiently transfected Expi293F cells. Co-transfection of the construct with a construct encoding Hu-C1r-LP-FLAG was performed to confirm cleavage of the nascent polypeptide. (Middle panel) Anti-Fc Western blot versus reducing SDS-PAGE of recombinant Fc-Hp beta fragment constructs made in transiently transfected Expi293F cells. (Right panel) Anti-Hp Western blot versus reducing SDS-PAGE of recombinant Hp beta fragment constructs made in transiently transfected Expi293F cells. (C) Analysis of aggregate content and protein processing by SEC and SDS-PAGE. (i) Analytical SEC chromatogram of Protein A affinity purified N-terminal extended Hu hemopexin-mIgG2aFc-Hu haptoglobin 2FS(148–406) / Hu-C1r-LP-FLAG performed on an Agilent 1260 Infinity system with a Superdex 200 Increase 5 / 150 column and MT-PBS mobile phase.(ii) Reducing and non-reducing SDS-PAGE gels (4-12% Bis-Tris) showing the purity and proper processing of Protein A affinity purified HuHemopexin-mIgG2aFc-HuHaptoglobin2FS(148-406). [Figure 10-2] Continued from Figure 10-1. [Figure 10-3] Continued from Figure 10-2. [Figure 10-4] Continued from Figure 10-3. [Figure 11-1] Qualitative Hb binding data based on size-exclusion HPLC chromatograms. The blue line represents the signal (405 nm) of the Hb+rHp mixture (at equimolar concentrations). (A) Huhaptoglobin(148-406)-8His, HSA-Huhaptoglobin(148-406)-8His, and muIgG2aFc-Huhaptoglobin(148-406)-8His. (B) Huhemopexin-Huhaptoglobin(148-406)-8His, Huhemopexin-msa-Huhaptoglobin2FS(148-406)-8His, and Huhemopexin-mIgG2aFc-Huhaptoglobin2FS(148-406)-His. Hemopexin was used as a negative control. The red line represents the signal of Hb alone, recorded at 405 nm. All size-exclusion HPLC traces were identically scaled to fit the red Hb peak chromatogram. [Figure 11-2] Continued from Figure 11-1. [Figure 12]Representative sensorgrams for each Hp variant analyzed for its ability to bind hemoglobin. Individual Hp variants were immobilized on the biosensor surface. After recording the baseline, hemoglobin across seven concentrations was probed (15, 7.5, 3.75, 1.88, 0.94, 0.47, and 0.32 nM). After subtracting the reference (assay buffer reference), the data was processed and globally fitted using a 1:1 binding model. The accuracy of the fit was described by Chi2 and R2. Hb is shown as the grey curve and the fitted curve is shown as a solid red line. (A) Hu haptoglobin 1-1. (B) Hu haptoglobin 2FS(148-406)-8His. (C) Hu hemopexin-Hu haptoglobin 2FS(148-406)-8His. [Figure 13] Figure 1 shows the heme binding capacity of different haptoglobin variants containing the hemopexin domain. The release of heme from heme-albumin in the presence of the different Hp variants indicated was measured by recording a series of UV-VIS spectra over time (5 hours at 37°C) for reaction mixtures containing 12.5 μM Hb(Fe3+) and 5 μM Hp protein (4 μM was used except for Huhemopexin-msa-Huhaptoglobin2FS(148-406)). Heme-albumin (blue curve) shows the concentration of heme bound to Hb at any given time point. Hemopexin:heme (red curve) shows the concentration of heme transferred from heme-albumin at any given time point. [Figure 14]Representative sensorgrams for each Hp variant analyzed for its ability to bind to the scavenger receptor CD163. Human CD163 receptor was immobilized on the biosensor surface. After recording a baseline, complexes spanning six concentrations were probed. After subtraction of the reference (see assay buffer), data were processed and globally fitted using a 1:1 binding model. The accuracy of the fit was described by Chi2 and R2. Hb is shown as the grey curve and the fitted curve is shown as a solid red line. In addition, KD was determined by steady-state analysis for the two recombinant variants. (A) Huhaptoglobin1-1:Hb complex (50-1.56 nM). (B) Huhaptoglobin2FS(148-406):Hb complex (2000-31.25 nM). (C) Hu hemopexin-Hu haptoglobin 2FS (148-406):Hb complex (1500-234.4 nM). [Figure 15] FIG. 1 shows vascular function, comparing rescue of NO-dependent vasodilation after addition of different Hp mutants. Vasodilatory responses to NO were measured after addition of Hb and then again after addition of Hb scavengers. The effects of Hp mutants (plasma Hp1-1, recHp1-1, recHpCD163low, miniHp, and SuperScavenger) were compared to the reference, Hp2-2 (blue; data set on the left of each window). [Figure 16-1] Figure 3: Comparison of the protective effect of different Hp variants on lipid peroxidation. (A) Fluorescence emission after 4 h of incubation at 37°C was used to measure the occurrence of MDA in mixtures of Hb with equimolar concentrations of Hp variants and rLP. Hb without scavenger proteins was used as a positive control and rLP alone was used as a negative control. (B) Different Hb scavengers (10 μM) and rLP (2 g / L) were incubated with a range of Hb concentrations (0-100 μM) at 37°C for 4 h. Lipid peroxidation was quantified using the TBARS assay. [Figure 16-2] Continued from Figure 16-1. [Figure 17-1] Binding affinity of (A) plasma-derived heme:Hx; and (B) heme:Hx-Hp complex and uncomplexed (inset) scavenger protein to biotinylated LRP1 cluster III. Grey curves represent sensorgrams for a range of ligand concentrations (all concentrations are 2000-31.25 nM) in the fluid phase. Fits are indicated by red curves. [Figure 17-2] Continued from Figure 17-1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0024] Unless otherwise specified, the indefinite articles "a," "an," and "the" as used herein include plural aspects. Thus, for example, reference to "an agent" includes a single agent, as well as two or more agents; reference to "a composition" includes a single composition, as well as two or more compositions; and so forth.

[0025] As used herein, the term "about" means ±10% of the recited value.

[0026] Unless the context requires otherwise, throughout this specification and the following claims, "comprise" and variations such as "comprises" and "comprising" shall be understood to imply the inclusion of a stated integer or step, or group of integers or steps, but not the exclusion of any other integer or step, or group of integers or steps.

[0027] The term "consisting of" means "consisting only of," which is inclusive and limited to that integer or step, or group of integers or steps, and excluding any other integer or step, or group of integers or steps.

[0028] The term "consisting essentially of" implies the inclusion of the stated integers or steps, or groups of integers or steps, but also includes other integers or steps, or groups of integers or steps, that do not materially alter or contribute to the operation of the invention.

[0029] Unless specified to the contrary, throughout this specification, references made to "%" content shall be understood to mean w / w (weight / weight) %. For example, a solution containing at least an 80% haptoglobin content, relative to total protein, shall be understood to mean a composition containing at least an 80% w / w haptoglobin content, relative to total protein.

[0030] As mentioned elsewhere herein, the present invention is based, at least in part, on the surprising discovery by the inventors that functional haptoglobin beta chain or hemoglobin-binding fragments thereof can be produced from N-terminally truncated prohaptoglobin (proHp) in a mammalian expression system. N-terminally truncated proHp can be advantageously modified to carry functional moieties such as Hpx, Fc, and albumin, thereby producing constructs with improved therapeutic properties. Furthermore, the inventors unexpectedly found that the expression system described herein advantageously results in stable transfection and expression of functional haptoglobin beta chain, which distinguishes it from existing expression systems that at best achieve transient transfection and generally cannot express functional Hp beta chain. The expression systems described herein also advantageously allow for the production of fusion proteins or conjugates, including those in which the fusion partner is positioned N-terminal to the β-chain fragment and conveniently linked to an intercysteine ​​residue via a disulfide bond.

[0031] Thus, in one aspect disclosed herein, there is provided an expression system for producing recombinant haptoglobin beta chain or a hemoglobin-binding fragment thereof in a mammalian cell, comprising: (a) a first nucleic acid sequence encoding an N-terminally truncated prohaptoglobin (proHp), the N-terminally truncated proHp comprising (i) at least 14 consecutive C-terminal amino acid residues of a haptoglobin alpha chain, and (ii) a haptoglobin beta chain, or a hemoglobin-binding fragment thereof, the N-terminally truncated proHp comprising an internal enzymatic cleavage site between the at least 14 consecutive C-terminal amino acid residues of the haptoglobin alpha chain and the haptoglobin beta chain, or a hemoglobin-binding fragment thereof; and (b) a second nucleic acid sequence encoding an enzyme capable of cleaving the N-terminal truncated proHp at the enzyme cleavage site; Includes; When the first and second nucleic acid sequences are introduced into a mammalian cell and the N-terminal truncated proHp and the enzyme are then expressed in the cell, the enzyme is capable of cleaving the N-terminal truncated proHp at the internal enzyme cleavage site, thereby releasing the haptoglobin beta chain or a hemoglobin-binding fragment thereof from the N-terminal truncated proHp. An expression system is provided.

[0032] N-terminally truncated prohaptoglobin Haptoglobin (Hp) is an abundant plasma protein that is primarily synthesized in the liver. Hp is a high affinity scavenger for free hemoglobin (Hb), which may be released from red blood cells during hemolysis. The complex formed between the two proteins (Hb:Hp complex) provides multiple protective activities that attenuate the toxic effects of free Hb in the kidney, blood vessels, and surrounding tissues where free Hb is accessible. The protection provided by Hp attenuates two major toxicological consequences of Hb. First, the large molecular size of the Hb:Hp complex prevents the extravasation of free Hb. This mechanism protects renal function and preserves nitric oxide (NO) homeostasis in blood vessels by limiting the access of free Hb to the blood vessel wall. Second, the formation of the Hb:Hp complex stabilizes the structure of the Hb molecule in a way that limits the transfer of heme from its globin chains to proteins and reactive lipids. These mechanisms largely contribute to the antioxidant function of Hp during hemolysis.Hp has also been shown to play a role in T cell immune responses, regulation of cell proliferation, angiogenesis, and arterial remodeling.

[0033] Hp is synthesized as a single polypeptide precursor, prohaptoglobin (proHp), which is proteolytically processed by the protease C1rLP (Krzysztof and Fries, PNAS, 2004, 101(40):14390-14395). Prohaptoglobin (proHp) is the primary translation product of Hp mRNA. In the endoplasmic reticulum, proHp dimerizes via disulfide bond formation and is proteolytically cleaved by C1r-LP (protease complement C1r subcomponent-like protein). As a result, Hp exists in most mammals as a 150 kDa dimeric protein consisting of two α light chains and two β heavy chains linked by a single disulfide bond (SS) between the two α chains. Most mammalian Hp proteins are composed of two (αβ) monomers linked together through an interface between two α chains resulting in an (αβ)2 structure (designated Hp1-1 in humans). In humans, three Hp phenotypes exist due to the presence of two alleles of the Hp gene, designated Hp1 and Hp2. The Hp2 allele, arising from an intragenic duplication of the Hp1 allele, encodes a slightly larger α chain but is otherwise identical to the Hp1 allele. The cysteine ​​residues connecting the α chains are duplicated in the encoded Hp2 protein, so the phenotypes Hp2-1 and Hp2-2 display a spectrum of diverse Hp(αβ) multimers. Haptoglobin-hemoglobin consists of a dimer of haptoglobin chains, each of which interacts with an αβ dimer of hemoglobin. At each end, the β-chains of haptoglobin form a stable complex with the hemoglobin dimer. Interaction with the clearance receptor CD163 is also mediated by the β-chains.

[0034] The primary function of Hp (i.e., binding to Hb and CD163) is mediated by a β-chain encoded by amino acid residues corresponding to amino acid residues 162-406 of human proHp, as shown in SEQ ID NO:1. However, recombinant expression of constructs encoding these amino acid sequences in mammalian cells does not result in expression of a protein product. Surprisingly, the inventors have now found that by introducing at least 14 additional amino acids N-terminal to the proteolytic cleavage site of proHp and co-expressing this construct with a serine protease, robust expression of the Hp β-chain that retains binding to Hb and CD163 is achieved. The inventors also surprisingly found that N-terminally truncated proHp can be modified by conjugating or linking the β-chain components of N-terminally truncated proHp to functional moieties such as Fc, albumin, or hemopexin (Hpx), still resulting in relatively high yields of the modified constructs, and noted that the functional moieties retain binding affinity for their respective targets, Hb and heme (and, in the case of Hpx fusion proteins, CD163 or CD91).

[0035] The term "N-terminally truncated proHp" shall be understood to mean a fragment of proHp having an amino acid sequence shorter than the length of the native (naturally occurring) proHp molecule due to the truncated N-terminus that would otherwise form part of the complete Hpα chain. ProHp may be truncated at its N-terminus by any number of amino acid residues, so long as the N-terminally truncated proHp retains at least 14 consecutive C-terminal amino acid residues of the Hpα chain. In an embodiment, the expressed N-terminally truncated proHp contains a disulfide bond between the 14 consecutive C-terminal amino acid residues of the Hpα chain and the Hpβ chain. In an embodiment, the N-terminally truncated proHp contains a disulfide bond between a cysteine ​​residue in the at least 14 consecutive C-terminal amino acid residues of the haptoglobin alpha chain and a cysteine ​​residue at a position corresponding to amino acid position 266 of SEQ ID NO:1. In an embodiment, the N-terminally truncated proHp contains cysteine ​​residues at positions corresponding to amino acid positions 149 and 266 of human proHp as shown in SEQ ID NO:1.

[0036] The present disclosure is not limited to N-terminally truncated proHp having a specific amino acid sequence or encoded by a specific nucleic acid sequence, but rather includes N-terminally truncated proHp: (i) at least 14 consecutive C-terminal amino acid residues of the haptoglobin alpha chain; (ii) a haptoglobin beta chain or a hemoglobin-binding fragment thereof; and (iii) an internal enzymatic cleavage site between at least 14 consecutive C-terminal amino acid residues of the haptoglobin alpha chain and the haptoglobin beta chain or a hemoglobin-binding fragment thereof; It is to be understood that any suitable N-terminally truncated proHp may be used in accordance with the present invention, provided that it appropriately contains:

[0037] Those of skill in the art will also be familiar with suitable amino acid sequences of the Hp alpha chain, illustrative examples of which include amino acid residues 19-160 of SEQ ID NO: 1, amino acid residues 19-100 of SEQ ID NO: 2, and amino acid residues 19-101 of SEQ ID NO: 3. In certain embodiments, the at least 14 contiguous C-terminal amino acid residues of the haptoglobin alpha chain comprise, consist of, or consist essentially of an amino acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to amino acid residues 148-161 of SEQ ID NO: 1 (i.e., VCGKPKNPANPVQR; SEQ ID NO: 8).

[0038] Those skilled in the art will also be familiar with suitable amino acid sequences of the Hpβ chain, which consist of the region of the Hpβ chain capable of binding to Hb and CD163, illustrative examples of which include amino acid residues 162 to 406 of SEQ ID NO:1 (human proHp isoform 1; proHp1), amino acid residues 102 to 340 of SEQ ID NO:2 (human proHp isoform 2; proHp2), and amino acid residues 103 to 343 of SEQ ID NO:3 (human proHp isoform 3; proHp3). In one embodiment, the Hpβ chain comprises, consists of, or consists essentially of an amino acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to amino acid residues 162-406 of SEQ ID NO:1. In one embodiment, the Hpβ chain comprises, consists of, or consists essentially of an amino acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to amino acid residues 102-340 of SEQ ID NO:2. In one embodiment, the Hpβ chain comprises, consists of, or consists essentially of an amino acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to amino acid residues 103-343 of SEQ ID NO:3. In one embodiment, the Hpβ chain comprises, consists of, or consists essentially of an amino acid sequence having at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to amino acid residues 162-406 of SEQ ID NO:1.In certain embodiments, the Hpβ chain comprises, consists of, or consists essentially of an amino acid sequence having at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to amino acid residues 102-340 of SEQ ID NO:2. In certain embodiments, the Hpβ chain comprises, consists of, or consists essentially of an amino acid sequence having at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to amino acid residues 103-343 of SEQ ID NO:3. In certain embodiments, the Hpβ chain comprises, consists of, or consists essentially of an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to amino acid residues 162-406 of SEQ ID NO: 1. In certain embodiments, the Hpβ chain comprises, consists of, or consists essentially of an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to amino acid residues 102-340 of SEQ ID NO:2. In one embodiment, the Hpβ chain comprises, consists of, or consists essentially of an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to amino acid residues 103 to 343 of SEQ ID NO:3.

[0039] In certain embodiments, the Hpβ chain comprises, consists of, or consists essentially of an amino acid sequence having at least 95% (e.g., 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to amino acid residues 162-406 of SEQ ID NO:1. In certain embodiments, the Hpβ chain comprises, consists of, or consists essentially of an amino acid sequence having at least 95% (e.g., 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to amino acid residues 102-340 of SEQ ID NO:2. In certain embodiments, the Hpβ chain comprises, consists of, or consists essentially of an amino acid sequence having at least 95% (e.g., 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to amino acid residues 103-343 of SEQ ID NO:3.

[0040] It will also be understood by those skilled in the art that in some cases the sequence of the N-terminally truncated proHp selected will depend on the intended use, including the intended therapeutic use. By way of example, when recombinant Hp is used for the treatment and / or prevention of a condition in a human subject, the amino acid sequence of the N-terminally truncated proHp will be advantageously derived from human proHp for reasons including minimizing the possibility that administration of recombinant Hp to a subject will generate antibodies against recombinant Hp that would reduce its efficacy in vivo if not derived from human proHp. Similarly, when recombinant Hp is used for the treatment and / or prevention of a condition in a non-human subject, such as for veterinary applications, the amino acid sequence of the N-terminally truncated proHp will be advantageously derived from a proHp isoform that is native to the non-human subject. Those skilled in the art will be familiar with suitable non-human isoforms of proHp, illustrative examples of which include canine, feline, equine, bovine, ovine, and primate proHp. Exemplary examples of primate proHp are described in GenBank Accession Nos. AFH32200 and JAB04820. In an embodiment, the N-terminally truncated proHp has an amino acid sequence derived from human N-terminally truncated proHp. Thus, in an embodiment, the haptoglobin is human haptoglobin. Those skilled in the art are familiar with suitable human proHp amino acid sequences, of which exemplary examples include the amino acid sequences of human proHp described in GenBank Accession Nos. NP_005134 (proHp, precursor of human Hp isoform 1; SEQ ID NO: 1; also referred to as isoform Hp1 or Hp1F), NP_001119574 (proHp, precursor of human Hp isoform 2; SEQ ID NO: 2; also referred to as isoform Hp2 or Hp2SS), and NP_001305067 (proHp, precursor of human Hp isoform 3; SEQ ID NO: 3; also referred to as isoform Hp3).Subtypes of human Hp isoforms are also known to those of skill in the art, illustrative examples of which include: (i) Hp1F (SEQ ID NO:1), which includes residues Asp and Lys at amino acid positions 70 and 71, respectively, as shown in SEQ ID NO:1; (ii) Hp1S, which includes residues Asn and Glu at positions corresponding to amino acid positions 70 and 71, respectively, of SEQ ID NO:1; (iii) Hp2SS (SEQ ID NO:2), which includes residues Asn and Glu at amino acid positions 70 and 71, respectively, and residues Asn and Glu at amino acid positions 129 and 130, respectively, as shown in SEQ ID NO:2; and (iv) Hp2FS, which includes residues Asp and Lys at positions corresponding to amino acid positions 70 and 71, respectively, and residues Asn and Glu at positions corresponding to amino acid positions 129 and 130, respectively, of SEQ ID NO:2.

[0041] In one embodiment, the haptoglobin is a human haptoglobin isoform, Hp1F, as described herein. In another embodiment, the haptoglobin is a human haptoglobin isoform, Hp1S, as described herein. In another embodiment, the haptoglobin is a human haptoglobin isoform, Hp2FS, as described herein. In another embodiment, the haptoglobin is a human haptoglobin isoform, Hp2SS, as described herein.

[0042] In certain embodiments, proHp comprises, consists of, or consists essentially of an amino acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 1. In certain embodiments, proHp comprises, consists of, or consists essentially of an amino acid sequence having at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 1. In certain embodiments, proHp comprises, consists of, or consists essentially of an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 1. In certain embodiments, proHp comprises, consists of, or consists essentially of an amino acid sequence having at least 95% (e.g., 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 1. In certain embodiments, proHp comprises, consists of, or consists essentially of an amino acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 2. In certain embodiments, proHp comprises, consists of, or consists essentially of an amino acid sequence having at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 2.In certain embodiments, proHp comprises, consists of, or consists essentially of an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 2. In certain embodiments, proHp comprises, consists of, or consists essentially of an amino acid sequence having at least 95% (e.g., 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 2. In certain embodiments, proHp comprises, consists of, or consists essentially of an amino acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 3. In certain embodiments, proHp comprises, consists of, or consists essentially of an amino acid sequence having at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 3. In certain embodiments, proHp comprises, consists of, or consists essentially of an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 3. In certain embodiments, proHp comprises, consists of, or consists essentially of an amino acid sequence having at least 95% (e.g., 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 3.

[0043] In one embodiment, N-terminally truncated proHp comprises, consists of, or consists essentially of an amino acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to amino acid residues 148-406 of SEQ ID NO:1. In certain embodiments, N-terminally truncated proHp comprises, consists of, or consists essentially of an amino acid sequence having at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to amino acid residues 148-406 of SEQ ID NO: 1. In certain embodiments, N-terminally truncated proHp comprises, consists of, or consists essentially of an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to amino acid residues 148-406 of SEQ ID NO: 1. In one embodiment, N-terminally truncated proHp comprises, consists of, or consists essentially of an amino acid sequence having at least 95% (e.g., 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to amino acid residues 148 to 406 of SEQ ID NO:1.

[0044] In one embodiment, N-terminally truncated proHp comprises, consists of, or consists essentially of an amino acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to amino acid residues 89 to 347 of SEQ ID NO:2. In certain embodiments, N-terminally truncated proHp comprises, consists of, or consists essentially of an amino acid sequence having at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to amino acid residues 89-347 of SEQ ID NO:2. In certain embodiments, N-terminally truncated proHp comprises, consists of, or consists essentially of an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to amino acid residues 89-347 of SEQ ID NO:2. In one embodiment, N-terminally truncated proHp comprises, consists of, or consists essentially of an amino acid sequence having at least 95% (e.g., 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to amino acid residues 89 to 347 of SEQ ID NO:2.

[0045] In one embodiment, N-terminally truncated proHp comprises, consists of, or consists essentially of an amino acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to amino acid residues 89 to 347 of SEQ ID NO:3. In certain embodiments, N-terminally truncated proHp comprises, consists of, or consists essentially of an amino acid sequence having at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to amino acid residues 89-347 of SEQ ID NO: 3. In certain embodiments, N-terminally truncated proHp comprises, consists of, or consists essentially of an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to amino acid residues 89-347 of SEQ ID NO: 3. In one embodiment, N-terminally truncated proHp comprises, consists of, or consists essentially of an amino acid sequence having at least 95% (e.g., 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to amino acid residues 89 to 347 of SEQ ID NO:3.

[0046] Reference to "at least 80%" includes, for example, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and 100% sequence identity after optimal alignment or best fit analysis. Optimal sequence alignment for aligning a comparison window may be performed by computer implementation of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0 by Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and best alignment (i.e., the alignment resulting in the highest percentage of homology over the comparison window) produced by any of a variety of selected methods. See also the BLAST family of programs disclosed, for example, by Altschul et al. (1997), Nucl. Acids. Res., 25: 3389. A detailed discussion of sequence analysis is found in Ausubel et al. (1994-1998), Current Protocols in Molecular Biology, John Wiley & Sons Inc, part 19.3.

[0047] As used herein, the term "sequence identity" refers to the degree to which sequences are identical or structurally similar on a nucleotide-by-nucleotide basis or amino acid-by-amino acid basis over a comparison window.Therefore, "sequence identity percentage" is calculated by, for example, comparing two optimally aligned sequences over a comparison window, determining the number of positions where identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occur in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to obtain the sequence identity percentage. For example, "sequence identity" is the "percentage match" calculated by the DNASIS computer program (Version 2.5 for Windows; available from Hitachi Software Engineering Co., Ltd., South San Francisco, California, USA) using the standard default values ​​used in the reference manual accompanying the software.

[0048] As used herein, the term "sequence identity" includes exact identity between sequences compared at the nucleotide or amino acid level. In this specification, the term is also used to include inexact identity (i.e., similarity) at the nucleotide or amino acid level, in which case any difference(s) between sequences are, however, any differences with respect to amino acids that are related to each other at the structural, functional, biochemical, and / or conformational levels (or any differences in the context of the nucleotide, the amino acid encoded by said nucleotide). For example, in the case of non-identity (similarity) at the amino acid level, "similarity" includes, however, amino acids that are related to each other at the structural, functional, biochemical, and / or conformational levels. In some embodiments, the comparison of nucleotides and sequences is made at the level of identity, not similarity. For example, leucine is substituted for an isoleucine or valine residue. This is referred to as a conservative substitution. In certain embodiments, the amino acid sequence is modified for the purpose of conservative substitution of any of the amino acid residues contained therein, such that the modification has no or negligible effect on the binding specificity or functional activity of the modified polypeptide as compared to the unmodified polypeptide.

[0049] Sequence identity as described herein typically relates to the percentage of amino acid residues in a candidate sequence that are identical to those of the corresponding peptide sequence, after aligning the sequences, introducing gaps, if necessary, to achieve the maximum percentage of homology, and leaving any conservative substitutions out of consideration as part of the sequence identity. Neither N-terminal nor C-terminal extensions nor insertions shall be considered as reducing sequence identity or sequence homology.

[0050] Also contemplated herein are functional variants of N-terminal truncated proHp. As used herein, the term "functional variant" refers to a peptide that shares at least some amino acid sequence identity with a native (naturally occurring) isoform of proHp (human proHp or non-human proHp) but retains the ability to bind to Hb. In this context of the present specification, the terms "functional variant" and "Hb-binding functional variant" are used interchangeably. The functional variant extends to a C-terminally truncated proHp (i.e., a C-terminally truncated Hp β chain), but it is understood that the C-terminally truncated proHp appropriately retains at least a portion of the Hb-binding region of the Hp β chain, as is familiar to those skilled in the art. Furthermore, those skilled in the art are familiar with suitable methods for screening for functional variants, including C-terminally truncated Hp β chains, that retain Hb-binding activity, exemplary examples of which are described elsewhere herein, such as surface plasmon resonance (SPR) and size exclusion chromatography (e.g., HPLC). These methods are also described in Schaer et al. (2018, BMC Biotechnol., 18:15), the contents of which are incorporated herein by reference.

[0051] The present disclosure also extends to functional variants that differ from the native sequence by one or more amino acid substitutions, including conservative amino acid substitutions, deletions, or insertions. In certain embodiments, the functional variants comprise an amino acid sequence that differs from the native sequence by conservative substitution of any of the amino acid residues contained therein, such that the modification has no or negligible effect on the binding specificity or functional activity of the functional variant to Hb when compared to the unmodified (e.g., native) molecule. Those skilled in the art are also familiar with suitable methods for screening for functional variants that contain one or more amino acid substitutions, deletions, or insertions and retain Hb binding activity, illustrative examples of which are described elsewhere herein.

[0052] In some embodiments, the functional variant comprises, consists of, or consists essentially of an amino acid sequence having at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 93%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, or preferably at least 99% sequence identity to amino acid residues 148-406 of SEQ ID NO:1, amino acid residues 89-347 of SEQ ID NO:2, or amino acid residues 89-347 of SEQ ID NO:3.

[0053] In certain embodiments, N-terminally truncated proHp comprises, consists of, or consists essentially of an amino acid sequence having at least 80% sequence identity with amino acid residues 148-406 of SEQ ID NO:1. In certain embodiments, N-terminally truncated proHp comprises, consists of, or consists essentially of an amino acid sequence having at least 90% sequence identity with amino acid residues 148-406 of SEQ ID NO:1. In certain embodiments, N-terminally truncated proHp comprises, consists of, or consists essentially of an amino acid sequence having at least 95% sequence identity with amino acid residues 148-406 of SEQ ID NO:1. In certain embodiments, N-terminally truncated proHp comprises, consists of, or consists essentially of amino acid residues 148-406 of SEQ ID NO:1.

[0054] In certain embodiments, the N-terminally truncated proHp comprises, consists of, or consists essentially of an amino acid sequence having at least 80% sequence identity with amino acid residues 89-347 of SEQ ID NO:2. In certain embodiments, the N-terminally truncated proHp comprises, consists of, or consists essentially of an amino acid sequence having at least 90% sequence identity with amino acid residues 89-347 of SEQ ID NO:2. In certain embodiments, the N-terminally truncated proHp comprises, consists of, or consists essentially of an amino acid sequence having at least 95% sequence identity with amino acid residues 89-347 of SEQ ID NO:2. In certain embodiments, the N-terminally truncated proHp comprises, consists of, or consists essentially of amino acid residues 89-347 of SEQ ID NO:2.

[0055] In certain embodiments, N-terminally truncated proHp comprises, consists of, or consists essentially of an amino acid sequence having at least 80% sequence identity with amino acid residues 89-347 of SEQ ID NO:3. In certain embodiments, N-terminally truncated proHp comprises, consists of, or consists essentially of an amino acid sequence having at least 90% sequence identity with amino acid residues 89-347 of SEQ ID NO:3. In certain embodiments, N-terminally truncated proHp comprises, consists of, or consists essentially of an amino acid sequence having at least 95% sequence identity with amino acid residues 89-347 of SEQ ID NO:3. In certain embodiments, N-terminally truncated proHp comprises, consists of, or consists essentially of amino acid residues 89-347 of SEQ ID NO:2.

[0056] In a preferred embodiment, the N-terminally truncated proHp comprises a naturally occurring internal enzymatic cleavage site between at least 14 consecutive C-terminal amino acid residues of the haptoglobin alpha chain and the haptoglobin beta chain or a hemoglobin-binding fragment thereof; i.e., the internal enzymatic cleavage site is natural to the proHp from which the amino acid sequence of the N-terminally truncated proHp is derived. In human proHp isoform 1 (SEQ ID NO: 1), the internal enzymatic cleavage site is located at amino acid positions 161 and 162 of SEQ ID NO: 1 such that enzymatic cleavage at this site separates the Hp alpha chain (amino acid residues 19-161 of SEQ ID NO: 1) from the Hp beta chain (amino acid residues 162-406 of SEQ ID NO: 1). In human proHp isoform 2 (SEQ ID NO:2), an internal enzymatic cleavage site is located at amino acid positions 162 and 163 of SEQ ID NO:1 such that enzymatic cleavage at this site separates the Hp alpha chain (amino acid residues 19-162 of SEQ ID NO:2) from the Hp beta chain (corresponding to amino acid residues 163-407 of SEQ ID NO:2). In human proHp isoform 3 (SEQ ID NO:3), an internal enzymatic cleavage site is located at amino acid positions 162 and 163 of SEQ ID NO:3 such that enzymatic cleavage at this site separates the Hp alpha chain (amino acid residues 19-162 of SEQ ID NO:3) from the Hp beta chain (corresponding to amino acid residues 163-407 of SEQ ID NO:3).

[0057] In other embodiments, the first nucleic acid sequence encoding the N-terminal truncated proHp may contain an internal enzyme cleavage site between at least 14 consecutive C-terminal amino acid residues of the haptoglobin alpha chain and the haptoglobin beta chain or a hemoglobin-binding fragment thereof; that is, the internal enzyme cleavage site is non-natural to the proHp from which the amino acid sequence of the N-terminal truncated proHp is derived. In this context, the internal enzyme cleavage site is selected so that it is compatible with the enzyme encoded by the second nucleic acid sequence of the expression system, so that the enzyme encoded by the second nucleic acid sequence is capable of cleaving the N-terminal truncated proHp at the non-natural enzyme cleavage site. Those skilled in the art will be familiar with suitable non-natural internal enzyme cleavage sites as well as their corresponding enzymes. Illustrative examples of suitable non-natural internal enzyme cleavage sites include furin cleavage sites, non-natural serine protease cleavage sites, cysteine ​​protease cleavage sites, aspartic acid protease cleavage sites, metalloprotease cleavage sites, and threonine protease cleavage sites. Thus, in certain embodiments disclosed herein, the internal enzyme cleavage site is selected from the group consisting of a furin cleavage site, a non-naturally occurring serine protease cleavage site, a cysteine ​​protease cleavage site, an aspartic protease cleavage site, a metalloprotease cleavage site, and a threonine protease cleavage site.

[0058] In certain embodiments, the internal enzymatic cleavage site is a cleavage site other than a serine protease. In a preferred embodiment, the serine protease cleavage site is a C1r-like protein (C1rLP) cleavage site or a functional variant thereof, as described elsewhere herein.

[0059] Further functional parts In the expression system disclosed herein, the N-terminally truncated proHp encoded by the first nucleic acid sequence may further comprise one or more additional functional moieties. In an embodiment, the functional moieties are linked, fused, conjugated, coupled, tethered, or otherwise attached to one or more of the at least 14 consecutive C-terminal amino acid residues of the haptoglobin alpha chain. In an embodiment, the additional functional moieties are linked, fused, conjugated, coupled, tethered, or otherwise attached to one or more of the amino acid residues of the haptoglobin beta chain. In an embodiment, the additional functional moieties are linked, fused, conjugated, coupled, tethered, or otherwise attached to the N-terminally truncated proHp by a disulfide bond at a cysteine ​​residue among the at least 14 consecutive C-terminal amino acid residues. In one embodiment, the further functional moiety is linked, fused, conjugated, coupled, tethered or otherwise joined to the N-terminally truncated proHp by a disulfide bond at a cysteine ​​residue corresponding to the amino acid position corresponding to 149 of SEQ ID NO:1.

[0060] In some embodiments, the functional moiety is covalently linked to the N-terminal truncated proHp. In other embodiments, the N-terminal truncated proHp is fused, coupled, or otherwise joined to one or more heterologous moieties as part of a fusion protein. The one or more additional functional moieties will suitably improve, enhance, or otherwise extend the activity and / or stability of the haptoglobin beta chain or hemoglobin-binding fragment thereof described herein.

[0061] To facilitate the isolation of the recombinant proteins described herein, fusion polypeptides are made in which the N-terminally truncated proHp or functional variants thereof are translationally fused (covalently linked) to a heterologous polypeptide that allows for isolation, such as by affinity chromatography. Suitable heterologous polypeptides are known to those skilled in the art, and illustrative examples thereof include His tags (e.g., 8 histidine residues), GST tags (glutathione-S-transferase), V5 tags, HA tags, CBP (chitin-binding protein) tags, MBP (maltose-binding protein) tags, streptavidin tags, SBP (streptavidin-binding protein), Myc tags, and biotin tags.

[0062] In some embodiments, the N-terminal truncated proHp described herein is suitably conjugated to a functional moiety for extending the half-life of recombinant haptoglobin beta chain or its hemoglobin-binding fragment in vivo.Those skilled in the art are familiar with suitable half-life extending functional moieties, illustrative examples of which include polyethylene glycol (PEGylation), glycosylated PEG, hydroxyethyl starch (HES), polysialic acid, elastin-like polypeptide, heparosan polymer, and hyaluronic acid.In some embodiments disclosed herein, the functional moiety is selected from the group consisting of polyethylene glycol (PEGylation), glycosylated PEG, hydroxyethyl starch (HES), polysialic acid, elastin-like polypeptide, heparosan polymer, and hyaluronic acid. The half-life-extending functional moiety is linked (e.g., fused, conjugated, tethered or otherwise attached) to the N-terminally truncated proHp or a functional variant thereof by any suitable means known to those skilled in the art, an illustrative example of which is via a chemical linker, as described, for example, in U.S. Pat. No. 7,256,253, the entire contents of which are incorporated herein by reference.

[0063] In other embodiments, the functional moiety is a half-life enhancing protein (HLEP). Those skilled in the art are familiar with suitable half-life enhancing proteins, of which illustrative examples include albumin and fragments thereof. Thus, in an embodiment, the HLEP is albumin or a fragment thereof. The N-terminus of albumin or a fragment thereof is linked, fused, conjugated, coupled, tethered, or otherwise joined to the C-terminus of the alpha and / or beta chains of the N-terminally truncated proHp. Alternatively or in addition, the C-terminus of albumin or a fragment thereof is linked, fused, conjugated, coupled, tethered, or otherwise joined to the N-terminus of the alpha and / or beta chains of the N-terminally truncated proHp. One or more HLEPs are fused to the N-terminal or C-terminal portion(s) of the alpha and / or beta chain of the N-terminally truncated proHp, provided that the recombinant haptoglobin beta chain or hemoglobin-binding fragment thereof does not abolish the ability of the recombinant haptoglobin beta chain or hemoglobin-binding fragment thereof to bind to cell-free Hb. However, it is understood that some reduction in the binding of the recombinant haptoglobin beta chain or hemoglobin-binding fragment thereof to cell-free Hb is acceptable, provided that it is still capable of forming a complex with and thereby neutralizing the cell-free Hb.

[0064] As used herein, the terms "human serum albumin" (HSA) and "human albumin" (HA) and "albumin" (ALB) are used interchangeably. The terms "albumin" and "serum albumin" are broader terms and include human serum albumin (and fragments and variants thereof), as well as albumins (and fragments and variants thereof) derived from other molecular species.

[0065] As used herein, "albumin" collectively refers to a polypeptide or amino acid sequence of albumin, or a fragment or variant of albumin, having one or more functional activities (e.g., biological activities) of albumin. In particular, "albumin" refers to human albumin or a fragment thereof, including the mature form of human albumin or albumin derived from other vertebrates, or a fragment thereof, or an analog or variant of these molecules or fragments thereof.

[0066] The fusion proteins described herein may suitably comprise naturally occurring polymorphic variants of human albumin and / or fragments of human albumin. Generally speaking, fragments or variants of albumin will be at least 10, preferably at least 40, or most preferably greater than 70 amino acids in length.

[0067] In some embodiments, the HLEP is an albumin variant that has enhanced binding to the FcRn receptor. Such albumin variants may result in an increase in the plasma half-life of Hp or its functional analogue compared to Hp or its functional fragment fused with wild-type albumin. The albumin portion of the fusion protein described herein may suitably comprise at least one subdomain or domain of human albumin, or a conservative modification thereof.

[0068] In some embodiments, a linker sequence is placed between the N-terminally truncated proHp and the functional moiety. The linker sequence may be a peptide linker consisting of one or more amino acids, in particular 1-50, preferably 1-30, preferably 1-20, preferably 1-15, preferably 1-10, preferably 1-5, or more preferably 1-3 (e.g. 1, 2, or 3) amino acids, which may be identical to each other or different from each other. Preferred amino acids present in said linker sequence include Gly and Ser. In a preferred embodiment, the linker sequence is substantially non-immunogenic to a subject treated according to the methods disclosed herein. By substantially non-immunogenic, it is meant that the linker sequence does not elicit a detectable antibody response against the linker sequence or the recombinant haptoglobin beta chain or hemoglobin-binding fragment thereof in a subject to which it is administered. A preferred linker is composed of alternating glycine and serine residues. The skilled artisan is familiar with suitable linkers, for which illustrative examples are described in WO2007 / 090584. In an embodiment, the peptide linker between the N-terminal truncated proHp and the functional moiety comprises, consists of, or essentially consists of peptide sequences that are used as natural interdomain linkers in human proteins. Such peptide sequences in their natural environment are located close to the protein surface and accessible to the immune system, so that they may assume natural tolerance to this sequence. Illustrative examples are given in WO2007 / 090584. Suitable cleavable linker sequences are described, for example, in WO2013 / 120939.

[0069] Illustrative examples of suitable HLEP sequences are described below. Also disclosed herein are fusions with the exact "N-terminal amino acid" or "C-terminal amino acid" of the respective HLEP, including deletion of one or more amino acids of the HLEP at the N-terminus, or fusions with the "N-terminal portion" or "C-terminal portion" of the respective HLEP. The fusion protein may contain more than one HLEP sequence, for example, two or three HLEP sequences. These multiple HLEP sequences are fused in tandem, for example as a series of repeats, to the C-terminal portion of the alpha and / or beta chain of Hp.

[0070] The HLEP portion of the fusion protein described herein may be a mutant of wild-type HELP. The term "mutant" when used in reference to the HELP portion of the fusion protein shall be understood to include conservative or non-conservative insertions, deletions, and / or substitutions, where such changes do not substantially alter the ability of recombinant haptoglobin beta chain or hemoglobin-binding fragments thereof to form complexes with and thereby neutralize cell-free Hb. The HLEP may suitably be derived from any vertebrate, particularly any mammal, such as human, monkey, cow, sheep, or pig. Non-mammalian HLEPs include, but are not limited to, hen and salmon HLEPs.

[0071] In one embodiment, the functional moiety is a half-life extending polypeptide. In one embodiment, the half-life extending polypeptide is selected from the group consisting of albumin, a member of the albumin family or a fragment thereof, hemopexin, a solvated random chain with a large hydrodynamic volume (e.g., XTEN (see Schellenberger et al., 2009, Nature Biotechnol. 27:1186-1190)), homoamino acid repeats (HAP) or proline-alanine-serine repeats (PAS), afamin, alpha-fetoprotein, vitamin D binding protein, transferrin or a variant or fragment thereof, the carboxyl terminal peptide (CTP) of the human chorionic gonadotropin β subunit, neonatal Fc receptor (FcRn), in particular a polypeptide capable of binding to immunoglobulin constant regions and portions thereof, e.g., Fc fragments, a polypeptide or lipid capable of binding to albumin, a member of the albumin family or a fragment thereof, or an immunoglobulin constant region or a portion thereof under physiological conditions. In certain embodiments, the immunoglobulin constant region or portion thereof is an Fc fragment of immunoglobulin G1 (IgG1), an Fc fragment of immunoglobulin G2 (IgG2), an Fc fragment of immunoglobulin A (IgA), or an Fc receptor binding fragment thereof. As used herein, a half-life enhancing polypeptide can be a full-length half-life enhancing protein, or one or more fragments thereof, that is capable of stabilizing or extending the therapeutic or biological activity of a recombinant haptoglobin beta chain or a hemoglobin-binding fragment thereof, in particular extending the in vivo half-life of a recombinant haptoglobin beta chain or a hemoglobin-binding fragment thereof.Such fragments may be fragments of 10 or more amino acids in length, may contain at least about 15, preferably at least about 20, preferably at least about 25, preferably at least about 30, preferably at least about 50, or more preferably at least about 100 or more consecutive amino acids from the HLEP sequence, or may contain some or all of the specific domains of the respective HLEP, so long as the HLEP fragment provides a functional half-life extension of at least 10%, preferably at least 20%, or more preferably at least 25%, compared to the respective Hp in the absence of the HLEP. Those skilled in the art are familiar with methods for determining whether a functional portion provides a functional half-life extension to a recombinant haptoglobin beta chain or hemoglobin-binding fragment thereof (in vivo or in vitro), illustrative examples of which are described elsewhere herein.

[0072] The conjugates and fusion proteins described herein are created by in-frame connection of at least two DNA sequences encoding N-terminal truncated proHp and one or more functional moieties, such as HLEP. Those skilled in the art will understand that translation of the DNA sequence encoding the conjugate or fusion protein results in a single peptide sequence. The in-frame insertion of the DNA sequence encoding the peptide linker according to the embodiments disclosed herein results in a conjugate or fusion protein comprising a recombinant haptoglobin beta chain or hemoglobin-binding fragment thereof, a suitable linker, and a functional moiety.

[0073] In certain embodiments disclosed herein, the functional moiety comprises, consists of, or essentially consists of a polypeptide selected from the group consisting of albumin or a fragment thereof, hemopexin, transferrin or a fragment thereof, the C-terminal peptide of human chorionic gonadotropin, XTEN sequence, homoamino acid repeat (HAP), proline-alanine-serine repeat (PAS), afamin, alpha-fetoprotein, vitamin D binding protein, a polypeptide capable of binding to albumin or immunoglobulin constant region under physiological conditions, neonatal Fc receptor (FcRn), in particular, a polypeptide capable of binding to immunoglobulin constant region and a portion thereof, preferably the Fc portion of an immunoglobulin, and any combination of the foregoing. In another embodiment, the functional moiety is selected from the group consisting of hydroxyethyl starch (HES), polyethylene glycol (PEG), polysialic acid (PSA), elastin-like polypeptide, heparosan polymer, hyaluronic acid and albumin binding ligand, e.g., fatty acid chain, and any combination of the foregoing.

[0074] In one embodiment, the functional moiety is hemopexin or a heme-binding fragment thereof. Hemopexin is a 61 kDa plasma beta-1B glycoprotein composed of a single peptide chain of 439 amino acids long formed by two four-bladed beta propeller domains resembling two thick disks interdigitated at a 90° angle and connected by an interdomain linker peptide. Heme released into the blood as a result of intravascular and extravascular hemolysis is bound between the two four-bladed beta propeller domains in a pocket formed by the interdomain linker peptide. Residues His213 and His266 coordinate the iron atom of heme, resulting in a stable bis-histidyl complex similar to hemoglobin. The term "heme-binding fragment" is to be understood as meaning a fragment of a native hemopexin molecule that contains a sufficient number of contiguous or non-contiguous amino acid residues of the native hemopexin molecule such that it retains at least a portion of its binding affinity to cell-free heme as the native molecule. Those skilled in the art are familiar with suitable methods for determining whether a fragment of hemopexin retains heme-binding activity, and illustrative examples are described elsewhere herein. In an embodiment, the heme-binding fragment of hemopexin comprises, consists of, or consists essentially of an amino acid sequence that has at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, or more preferably at least 95% sequence identity to the native hemopexin protein. In an embodiment, the hemopexin is human hemopexin. In one embodiment, the human hemopexin comprises, consists of, or consists essentially of the amino acid sequence set forth in NP_000604 (SEQ ID NO: 12).

[0075] Hemopexin contains about 20% carbohydrate, including sialic acid, mannose, galactose, and glucosamine. Twelve cysteine ​​residues, which presumably account for six disulfide bridges, were found within the protein sequence. Hemopexin has a high affinity (K d Hemopexin represents the first line of defense against heme toxicity due to its ability to bind heme at <1 pM) and function as a heme-specific carrier from the bloodstream to the liver. Hemopexin binds heme in equimolar ratios, but there is no evidence that heme is covalently bound to proteins. In addition to binding to heme, hemopexin preparations have also been reported to possess several other functions, such as serine protease activity (Lin et al., 2016, Molecular Medicine, 22:22-31), as well as anti- and pro-inflammatory activities, inhibition of cell adhesion, and binding to certain divalent metal ions. While endogenous hemopexin can control the deleterious effects of free heme under physiological steady-state conditions, it has little effect on maintaining steady-state heme levels under pathophysiological conditions, such as those associated with hemolysis, where high levels of heme lead to depletion of endogenous hemopexin and cause heme-mediated oxidative tissue damage. Studies have shown that hemopexin infusion attenuates heme-induced endothelial activation, inflammation, and oxidative damage in experimental mouse models of hemolytic disorders such as sickle cell disease (SCD) and β-thalassemia. Administration of hemopexin has also been shown to significantly reduce levels of pro-inflammatory cytokines and counter heme-induced vasoconstriction in hemolytic animals.

[0076] In an embodiment, the functional moiety is an immunoglobulin molecule, including an Fc region or an FcRn-binding fragment thereof. Immunoglobulin Fc regions (Fc) are known in the art to extend the half-life of therapeutic proteins (see, for example, Dumont JA et al., 2006. BioDrugs, 20:151-160). The IgG constant region of the heavy chain consists of three domains (CH1-CH3) and a hinge region. The immunoglobulin sequence may be from any mammal or from each of the subclasses IgG1, IgG2, IgG3, or IgG4. IgG and IgG fragments without antigen-binding domains are also used as functional moieties, including when used as HLEPs. Hp or functional analogs thereof may be suitably connected to IgG or IgG fragments via the hinge region of the antibody or via a peptide linker, which may be cleavable. Some patents and patent applications describe the fusion of therapeutic proteins to immunoglobulin constant regions, which enhances the in vivo half-life of therapeutic proteins.For example, US2004 / 0087778 and WO2005 / 001025 describe fusion proteins of at least a portion of Fc domain or immunoglobulin constant region with biologically active peptides, which extend the half-life of peptides that are otherwise rapidly eliminated in vivo.Fc-IFN-β fusion proteins have been described to achieve enhanced biological activity, extended circulating half-life, and increased solubility (WO2006 / 000448A2). In addition to Fc-EPO proteins with extended serum half-lives and increased potency in vivo (WO 2005 / 063808 A1), Fc fusions with G-CSF (WO 2003 / 076567 A2), glucagon-like peptide 1 (WO 2005 / 000892 A2), coagulation factor (WO 2004 / 101740 A2), and interleukin 10 (U.S. Patent No. 6,403,077), all with half-life enhancing properties, have also been disclosed.

[0077] Illustrative examples of suitable HLEPs for use in accordance with the present invention are also described in WO2013 / 120939A1, the contents of which are incorporated herein by reference in their entirety.

[0078] Expression system As mentioned elsewhere herein, the present disclosure provides: (a) a first nucleic acid sequence encoding an N-terminally truncated prohaptoglobin (proHp), the N-terminally truncated proHp comprising (i) at least 14 consecutive C-terminal amino acid residues of a haptoglobin alpha chain, and (ii) a haptoglobin beta chain, or a hemoglobin-binding fragment thereof, the N-terminally truncated proHp comprising an internal enzymatic cleavage site between the at least 14 consecutive C-terminal amino acid residues of the haptoglobin alpha chain and the haptoglobin beta chain, or a hemoglobin-binding fragment thereof; and (b) a second nucleic acid sequence encoding an enzyme capable of cleaving the N-terminal truncated proHp at the enzyme cleavage site; Includes; The first and second nucleic acid sequences are introduced into a mammalian cell, and thereafter, in the cell, the N-terminal truncated proHp and the enzyme are expressed, whereby the enzyme is capable of cleaving the N-terminal truncated proHp at the internal enzyme cleavage site, thereby releasing the haptoglobin beta chain or a hemoglobin-binding fragment thereof from the N-terminal truncated proHp. A mammalian expression system is presented.

[0079] The expression system described herein is advantageously based on mammalian cells, since it can produce recombinant proteins that are likely to maintain biological activity, for example by facilitating the proper folding and post-translational modification of proteins required to preserve function within the expressed protein(s).As mentioned elsewhere herein, the inventors have unexpectedly found that their expression system is advantageous because it results in stable transfection and expression of functional haptoglobin beta chain, which distinguishes them from existing expression systems that at best achieve transient transfection and generally cannot produce functional proteins.Thus, the expression system described herein is capable of stable transfection and expression of functional recombinant haptoglobin beta chain or hemoglobin-binding fragment thereof in mammalian cells. The skilled artisan is familiar with suitable methods for preparing recombinant proteins, illustrative examples of which include the introduction of one or more nucleic acid molecules comprising one or more nucleic acid sequences encoding the desired recombinant protein described herein into a suitable host cell capable of expressing said nucleic acid sequences, incubating said host cell under conditions suitable for the expression of said nucleic acid sequences, and recovering said recombinant protein.

[0080] Based on knowledge of the genetic code, suitable methods for preparing nucleic acid molecules encoding recombinant proteins, possibly including a step of optimizing codons based on the nature of the host cells (e.g., human cells and non-human mammalian cells) used to express and / or secrete the recombinant fusion protein, will also be known to those skilled in the art. Mammalian cells suitable for expressing recombinant proteins are also known to those skilled in the art, illustrative examples of which include Chinese hamster ovary (CHO) cells and their derivatives (e.g., CHO-K1 cells and CHO pro-3 cells), mouse myeloma cells (e.g., NS0 cells and Sp2 / 0 cells), human embryonic kidney cells (e.g., HEK293 cells). Protein expression in mammalian cells is also achieved using virus-mediated transduction by techniques such as the BacMam system. This technique utilizes recombinant baculoviruses for simple transduction of mammalian cells, allowing the production of milligram quantities of proteins for structural studies. Other cell lines, such as COS and Vero cells (both African Green Monkey Kidney cells), HeLa (human cervical carcinoma), and NS0 (mouse myeloma), have also been used for structural studies. Some of these cell lines, such as NS0, are more difficult to transfect. Transfection is usually achieved using electroporation and is only used to generate stable cell lines. Illustrative examples of mammalian cells suitable for expression of recombinant proteins, including those described herein, are described in Khan KH (2013, Adv. Pharm. Bull., 3(2):257-263), such as U2OS cells, A549 cells, HT1080 cells, CAD cells, P19 cells, NIH3T3 cells, L929 cells, N2a cells, human embryonic kidney 293 cells, HEK293T cells, Chinese hamster ovary cell line, MCF-7, Y79 cells, SO-Rb50 cells, HepG2 cells, DUKX-X11 cells, J558L cells, and baby hamster kidney (BHK) cells.

[0081] Also see "Short Protocols in Molecular Biology, 5th ed., 2 vols., A Compendium of Methods from Current Protocols in Molecular Biology" (Frederick M. Ausubel (ed.), Roger Brent (ed.), Robert E. Kingston (ed.), David D. Moore (ed.), JG Seidman (ed.), John A. Smith (ed.), Kevin Struhl (ed.), J Wiley & Sons, London).

[0082] In one embodiment of the present invention, a mammalian cell is provided comprising a first nucleic acid sequence and a second nucleic acid sequence according to the present invention, wherein the mammal is capable of expressing an N-terminal truncated proHp and a serine protease in the cell, the serine protease being capable of cleaving the N-terminal truncated proHp within the C1rLP cleavage site, thereby releasing the haptoglobin beta chain or a hemoglobin-binding fragment thereof from the N-terminal truncated proHp of the present invention. Suitable mammalian cells are known to those skilled in the art, and illustrative examples thereof include CHO cells, COS-7 cells, Vero cells, NIH3T3 cells, L929 cells, N2a cells, BHK cells, mouse ES cells, and human cells such as HeLa cells, HEK-293 cells, HEK-293T cells, U20S cells, A549 cells, HT1080 cells, WI-38 cells, MRC-5 cells, Namalwa cells, HepG2 cells, etc.

[0083] As used herein, terms such as "encoding", "encoding" and the like refer to the ability of a nucleic acid to result in another nucleic acid or a polypeptide. For example, a nucleic acid sequence is said to "encode" a polypeptide when it is transcribed and / or translated to produce a polypeptide, or is transcribed and / or translated to produce a polypeptide, typically in a host cell. Such a nucleic acid sequence may include coding sequences, or both coding and non-coding sequences. Thus, terms such as "encoding", "encoding" and the like include an RNA product resulting from transcription of a DNA molecule, a protein resulting from translation of an RNA molecule, a protein resulting from transcription of a DNA molecule to form an RNA product and then translation of the RNA product, or a protein resulting from transcription of a DNA molecule to result in an RNA product, processing of the RNA product to result in a processed RNA product (e.g., mRNA), and then translation of the processed RNA product. In some embodiments, a nucleic acid sequence encoding a peptide sequence described herein, or a fusion protein described herein, is codon-optimized for expression in a suitable host cell. For example, if the recombinant protein is used to treat or prevent a condition associated with cell-free hemoglobin (Hb) in a human subject, the nucleic acid sequence can be a codon-optimized human nucleic acid sequence. Suitable methods for codon optimization will be known to those of skill in the art, such as using the "Reverse Translation" option of the "Gene Design" tool located within "Software Tools" on the John Hopkins University Build a Genome website.

[0084] As mentioned elsewhere herein, the sequences are linked to each other in the recombinant protein by any means known to those skilled in the art. The terms "link" and "linked" include the direct linkage of two sequences (e.g., peptide sequences) via a peptide bond; i.e., the C-terminus of one sequence is covalently linked to the N-terminus of another sequence via a peptide bond. The terms "link" and "linked" also include within their meaning the linkage of two sequences (e.g., peptide sequences) via interspersed linker elements.

[0085] Isolation and cloning of nucleic acid sequences are accomplished using standard techniques (see, e.g., Ausubel et al., supra). For example, any desired nucleic acid sequence is obtained directly from the virus by extracting RNA and then synthesizing cDNA from the RNA template (e.g., by RT-PCR) by standard techniques. The nucleic acid sequence is then inserted into a suitable expression vector, either directly or after one or more subcloning steps. Those skilled in the art will understand that the details of the exact vector used are not critical. Illustrative examples for suitable vectors include plasmids, phagemids, cosmids, bacteriophages, baculoviruses, retroviruses, or DNA viruses. The desired recombinant protein(s) are then expressed and purified, as described in more detail below. Alternatively, the nucleic acid sequence is further manipulated to introduce one or more mutations, such as those described above, by standard in vitro site-directed mutagenesis methods known to those skilled in the art. Mutations are introduced by deletion, insertion, substitution, inversion, or combination of one or more of the appropriate nucleotides that make up the coding sequence. This is achieved, for example, by PCR-based techniques, for which primers are designed and incorporate one or more mismatches, insertions, or deletions of nucleotides. The presence of mutations is verified by a number of standard techniques, for example, restriction analysis or DNA sequencing. Methods for producing recombinant proteins are well known to those skilled in the art. The DNA sequence that codes for the recombinant protein is inserted into a suitable expression vector, the selection of which is known to those skilled in the art. Suitable examples of expression vectors include, but are not limited to, the following expression vectors:When recombinant protein(s) are expressed in mammalian cells such as CHO cells, COS cells, and NIH3T3 cells, the expression vector contains a promoter necessary for expression in these cells, such as the SV40 promoter (Mulligan et al., Nature, 277:108 (1979)) (e.g., early simian virus 40 promoter), MMLV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res., 18:5322 (1990)), or CMV promoter (e.g., human cytomegalovirus immediate early promoter). The recombinant expression vector may also carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origin of replication) and selectable marker genes. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Examples of vectors with selectable markers include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

[0086] It will be understood that the expression vector may further comprise regulatory elements, such as transcriptional elements required for efficient transcription of the DNA sequence encoding the coat protein or fusion protein. Illustrative examples of suitable regulatory elements to be incorporated into the vector include promoters, enhancers, terminators, and polyadenylation signals (e.g., derived from SV40, CMV, adenovirus, etc., such as the CMV enhancer / AdMLP promoter regulatory element, or the SV40 enhancer / AdMLP promoter regulatory element, selected according to the host cell) that drive high levels of transcription of the nucleic acid.

[0087] In certain embodiments, the nucleic acids described herein are incorporated into a nucleic acid cassette, also referred to herein as an expression cassette. Nucleic acid cassette or expression cassette is intended to mean a nucleic acid sequence designed to introduce a nucleic acid sequence, typically a heterologous nucleic acid sequence (e.g., described herein as a nucleic acid construct), into a vector. The expression cassette may contain terminal restriction enzyme linkers (i.e., restriction enzyme recognition nucleotides) at each end of the cassette sequence to facilitate the insertion of the nucleic acid sequence(s) of interest. The terminal restriction enzyme linkers at each end may be the same terminal restriction enzyme linker or different terminal restriction enzyme linkers. In some embodiments, the terminal restriction enzyme linkers may contain rare restriction enzyme recognition / cleavage sequences to prevent unintended digestion of the nucleic acid or alphavirus genome into which the cassette is introduced. Suitable terminal restriction enzyme linkers will be known to those of skill in the art. In one embodiment, the restriction enzyme recognition nucleotides for Pac I (TTAATTAA) are added to the 5' end of each expression cassette, and the restriction enzyme recognition nucleotides for Sbf I (CCTGCAGG) are added to the 3' end of each expression cassette.

[0088] In certain embodiments, the transcriptional and translational regulatory control sequences include nucleotide sequences encoding promoter sequences, 5' non-coding regions, cis regulatory regions such as functional binding sites for transcriptional or translational regulatory proteins, an upstream open reading frame, an internal ribosome entry site (IRES), a transcription initiation site, a translation initiation site, and / or a leader sequence, a stop codon, a translation stop site, and a 3' non-translated region.

[0089] In an embodiment, the first nucleic acid and the second nucleic acid are cloned into the same expression cassette and under the control of separate promoters. In another embodiment, the first nucleic acid and the second nucleic acid are cloned into the same expression cassette and under the control of the same promoter. In this context, a single promoter drives the expression of two open reading frames. In another embodiment, the first nucleic acid and the second nucleic acid are cloned into separate expression cassettes. It is to be understood that the expression system described herein is advantageous in some contexts because it comprises each of the first and second nucleic acid sequences in separate expression vectors. Thus, in an embodiment, the expression system comprises (i) a first expression vector comprising the first nucleic acid sequence described herein, and (ii) a second expression vector comprising the second nucleic acid sequence described herein.

[0090] The expression cassettes contemplated herein may also include one or more selectable marker sequences suitable for use in identifying host cells that have been infected, transformed, or transfected with the expression cassette, or host cells that have not been infected, transformed, or transfected with the expression cassette. Markers include, for example, genes encoding proteins that increase or decrease resistance or sensitivity to antibiotics or other compounds, genes encoding enzymes whose activities are detectable by standard assays known in the art (e.g., β-galactosidase, luciferase), and genes that visibly affect the phenotype of transformed or transfected cells, hosts, colonies, or plaques that carry the expression cassette (e.g., various fluorescent proteins, such as green fluorescent protein GFP).

[0091] The present disclosure also extends to host cells which contain the polynucleotide compositions described herein.

[0092] As used herein, a host cell is understood to mean a cell that contains the polynucleotide composition described herein. The host cell can be a bacterial cell, a yeast cell, an insect cell line, or a mammalian cell line. In a preferred embodiment, the host cell is a cell within the subject to which the polynucleotide composition described herein is administered.

[0093] The host cells may be transfected and / or infected with the vectors, and their progeny, so as to be capable of expressing the polynucleotide compositions described herein and producing the recombinant proteins described herein.

[0094] Suitable host cell lines are known to those skilled in the art and are commercially available, for example, through established cell culture collections. Such cells are then used to produce recombinant proHp or for other uses as required. An exemplary method may include culturing cells containing the polynucleotide composition (e.g., optionally under the control of an expression sequence) under cell culture conditions that allow optimal production of recombinant protein, and then isolating the recombinant protein from the cells or cell culture medium using standard techniques known to those skilled in the art. The present disclosure also extends to recombinant proteins isolated from cultured mammalian cells modified to express N-terminally truncated proHp as described herein, including recombinant haptoglobin beta chain and hemoglobin-binding fragments thereof, as well as any of one or more functional portions described elsewhere herein.

[0095] The expression system according to the present invention comprises a first nucleic acid sequence encoding an N-terminally truncated proHp comprising at least 14 consecutive C-terminal amino acid residues of the haptoglobin alpha chain and a haptoglobin beta chain or a hemoglobin-binding fragment thereof. The hemoglobin-binding fragment of the haptoglobin beta chain may be of any suitable length, provided that the fragment retains the ability to form a complex with cell-free Hb and thereby neutralize its biological activity. The N-terminally truncated proHp further comprises an internal C1r-like protein (C1rLP) cleavage site between the at least 14 consecutive C-terminal amino acid residues of the haptoglobin alpha chain and the haptoglobin beta chain or a hemoglobin-binding fragment thereof. In a preferred embodiment, the at least 14 consecutive C-terminal amino acid residues of the haptoglobin alpha chain comprise at least one cysteine ​​residue. In an embodiment, the cysteine ​​residue is located at amino acid position 14 of the at least 14 consecutive C-terminal amino acid residues of the haptoglobin alpha chain. Advantageously, a cysteine ​​residue within the at least 14 consecutive C-terminal amino acid residues of the haptoglobin alpha chain can form a disulfide bond with a cysteine ​​residue of the beta chain that would otherwise be free, which can aid in the expression and / or purification of a recombinant haptoglobin beta chain or a hemoglobin-binding fragment thereof.

[0096] In some embodiments, the expression of the N-terminally truncated proHp in the mammalian cell is driven by a first mammalian regulatory sequence operably linked to a first nucleic acid sequence, and the expression of the serine protease in the mammalian cell is driven by a second mammalian regulatory sequence operably linked to a second nucleic acid sequence. The first mammalian regulatory sequence may be the same sequence as the second mammalian regulatory sequence or may be a different sequence. In some embodiments, the first mammalian regulatory sequence is different from the second mammalian regulatory sequence.

[0097] The present disclosure also extends to an expression vector for producing recombinant haptoglobin beta chain or hemoglobin-binding fragment thereof in a mammalian cell as described herein. The vector may include a first nucleic acid sequence as described herein and a second nucleic acid sequence as described herein. The first nucleic acid and the second nucleic acid may be operably linked to the same mammalian regulatory sequence or may be operably linked to different mammalian regulatory sequences. Thus, in some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are operably linked to a common mammalian regulatory sequence. In other embodiments, the first nucleic acid sequence is operably linked to a first mammalian regulatory sequence and the second nucleic acid sequence is operably linked to a second mammalian regulatory sequence, where the first mammalian regulatory sequence is different from the second mammalian regulatory sequence. As mentioned elsewhere herein, the present disclosure also extends to an expression system comprising (i) a first expression vector comprising a first nucleic acid sequence as described herein, and (ii) a second vector comprising a second nucleic acid sequence as described herein, each of the first and second nucleic acid sequences being operably linked to a regulatory sequence, preferably a mammalian regulatory sequence, as described herein.

[0098] The present disclosure also extends to a method of producing a recombinant haptoglobin beta chain or hemoglobin-binding fragment thereof, comprising introducing into a mammalian cell an expression system as described herein or an expression vector(s) as described herein. A person skilled in the art will be familiar with suitable methods of introducing an expression system or expression vector(s) into a mammalian cell. For example, biological transfection methods (e.g., viral-mediated transfection methods), chemical transfection methods (e.g., cationic polymer, calcium phosphate, cationic lipid, or cationic amino acid transfection methods) or physical transfection methods (e.g., direct injection, biolistic particle delivery, electroporation, laser irradiation, sonoporation, or magnetic nanoparticle transfection methods) are utilized. In certain embodiments, the introduction of the expression system or expression vector(s) into a mammalian cell is accomplished using a cationic, lipid-based transfection reagent.

[0099] When the expression system or vector(s) described herein is introduced into a mammalian cell and the cell is cultured under suitable conditions, the N-terminal truncated proHp and the serine protease are expressed in the cell. Without being limited to theory or a particular application mode, it is understood that upon expression, the expressed serine protease cleaves the expressed N-terminal truncated proHp within the C1rLP cleavage site, releasing the haptoglobin beta chain or its hemoglobin-binding fragment from the N-terminal truncated proHp. The cell may be cultured under conditions and for a time sufficient to allow the production of recombinant haptoglobin beta chain or its hemoglobin-binding fragment. Those skilled in the art are familiar with suitable culture conditions and culture media, including commercially available cell culture media, illustrative examples of which are described, for example, in Laurenti and Ooi (2013, 998:10.1007 / 978-1-62703-351-0_2, "Methods in Molecular biology" (Clifton, NJ); and Kaufman RJ (2000, Mol. Biotechnol., 16:151-160). Note that the kinetics of recombinant protein expression may vary between mammalian cell types, and that culture conditions and times sufficient to allow suitable expression of recombinant proteins in mammalian cells having the expression systems disclosed herein depend on the type of mammalian cell(s) utilized. In any event, culture conditions and times are optimized by routine experimentation.

[0100] Non-limiting examples of suitable mammalian cells are described elsewhere herein and include human cells, bovine cells, ovine cells, equine cells, goat cells, rabbit cells, guinea pig cells, rat cells, hamster cells, or mouse cells, HEK293 (human embryonic kidney) cells, CHO (Chinese hamster ovary) cells, and mouse myeloma cells. Other illustrative examples of suitable mammalian cells include HeLa cells, HEK293T cells, U2OS cells, A549 cells, HT1080 cells, CAD cells, P19 cells, NIH3T3 cells, L929 cells, N2a cells, HEK293 cells, CHO cells, MCF-7 cells, Y79 cells, SO-Rb50 cells, HepG2 cells, DUKX-X11 cells, J558L cells, and BHK cells. In certain embodiments, the mammalian cells are human cells. In another embodiment, the mammalian cell is a human fetal cell, preferably a human fetal kidney cell (eg, HEK293).

[0101] The present invention further provides mammalian cells modified to carry the expression systems described herein.

[0102] The recombinant haptoglobin beta chain, or hemoglobin-binding fragment thereof, is isolated and purified using any suitable method known in the art. In some cases, purification is performed by chromatography, such as tandem chromatography, as illustrated in the Examples.

[0103] An enzyme encoded by a second nucleic acid sequence As mentioned elsewhere herein, the expression system disclosed herein comprises a second nucleic acid sequence encoding an enzyme capable of cleaving the N-terminal truncated proHp encoded by the first nucleic acid sequence at the enzyme cleavage site described herein. It will therefore be understood that the selection of the enzyme encoded by the second nucleic acid sequence will depend on the internal enzyme cleavage site between at least 14 consecutive C-terminal amino acid residues of the haptoglobin alpha chain and the haptoglobin beta chain or a hemoglobin-binding fragment thereof of the N-terminal truncated proHp; that is, the enzyme encoded by the second nucleic acid sequence will be compatible with the internal enzyme cleavage site so that it can properly cleave the N-terminal truncated proHp at the enzyme cleavage site when the first and second nucleic acid sequences are expressed in a mammalian cell. Those skilled in the art are familiar with suitable internal enzyme cleavage sites, illustrative examples of which are described elsewhere herein, such as furin cleavage sites, non-natural serine protease cleavage sites, cysteine ​​protease cleavage sites, aspartic acid protease cleavage sites, metalloprotease cleavage sites, and threonine protease cleavage sites.In an embodiment, the enzyme encoded by the second nucleic acid sequence of the expression system described herein is selected from the group consisting of furin, serine protease, cysteine ​​protease, aspartic acid protease, metalloprotease, and threonine protease.In an embodiment, the enzyme encoded by the second nucleic acid sequence of the expression system described herein is a serine protease.

[0104] The skilled artisan is familiar with suitable serine proteases, illustrative examples of which are described, for example, in Di Cera (IUBMB Life, 2009, 61(5):510-515). In one embodiment, the serine protease is C1rLP, a C1r-like serine protease, or a functional variant thereof. The term "functional variant" as used in reference to C1rLP shall be understood to include a serine protease having an amino acid sequence that differs from its native counterpart by one or more amino acid substitutions, deletions, and / or insertions, including conservative or non-conservative amino acid substitutions, such that such differences do not substantially alter the ability of the variant to cleave N-terminally truncated proHp at the internal C1rLP cleavage site. The functional variant may be a naturally occurring functional variant or a recombinant or synthetic (e.g., chemically synthesized) functional variant using methods known to those skilled in the art. Functional variants of C1rLP extend to naturally occurring isoforms, examples of which are known to those of skill in the art, such as C1rLP isoform 1 (e.g., GenBank Accession No.: NP_057630; SEQ ID NO: 4), C1rLP isoform 2 (e.g., GenBank Accession No.: NP_001284569; SEQ ID NO: 5), C1rLP isoform 3 (e.g., GenBank Accession No.: NP_001284571; SEQ ID NO: 6), and C1rLP isoform 4 (e.g., GenBank Accession No.: NP_001284572; SEQ ID NO: 7). In an embodiment, the serine protease C1rLP comprises, consists of, or consists essentially of the amino acid sequence of any one of SEQ ID NOs: 4-7. In an embodiment, the serine protease or functional variant thereof comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 4. In another embodiment, the serine protease or functional variant thereof comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 5. In another embodiment, the serine protease or functional variant thereof comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 6.In another embodiment, the serine protease or functional variant thereof comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 7. In one embodiment, the serine protease or functional variant thereof is C1rLP comprising, consisting of, or consisting essentially of an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99%, or preferably 100% sequence identity to any one of SEQ ID NOs: 4-7, e.g., after optimal alignment or best fit analysis. In one embodiment, the serine protease or functional variant thereof is C1rLP comprising, consisting of, or consisting essentially of an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99%, or preferably 100% sequence identity to SEQ ID NO:4, e.g., after optimal alignment or best fit analysis.In one embodiment, the serine protease or functional variant thereof is C1rLP comprising, consisting of, or consisting essentially of an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99%, or preferably 100% sequence identity to SEQ ID NO:5, e.g., after optimal alignment or best fit analysis. In one embodiment, the serine protease or functional variant thereof is C1rLP comprising, consisting of, or consisting essentially of an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99%, or preferably 100% sequence identity to SEQ ID NO:6, e.g., after optimal alignment or best fit analysis.In one embodiment, the serine protease or functional variant thereof is C1rLP comprising, consisting of, or consisting essentially of an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99%, or preferably 100% sequence identity to SEQ ID NO:7, e.g., after optimal alignment or best fit analysis.

[0105] Pharmaceutical compositions and uses thereof The present disclosure also extends to a pharmaceutical composition comprising a therapeutically effective amount of a recombinant haptoglobin beta chain or a hemoglobin-binding fragment thereof prepared according to the methods described herein, and optionally a pharma- ceutically acceptable carrier.

[0106] The present disclosure also extends to recombinant hemoglobin-binding molecules comprising (i) a haptoglobin beta chain or a hemoglobin-binding fragment thereof, and (ii) an N-terminally truncated haptoglobin alpha chain, wherein the N-terminally truncated haptoglobin alpha chain comprises at least 14 contiguous C-terminal amino acid residues of the haptoglobin alpha chain, wherein the at least 14 contiguous C-terminal amino acid residues of the haptoglobin alpha chain are non-contiguous with the haptoglobin beta chain or a hemoglobin-binding fragment thereof, and wherein the N-terminally truncated haptoglobin alpha chain is joined to the haptoglobin beta chain or a hemoglobin-binding fragment thereof. By "non-contiguous" it is meant that the recombinant hemoglobin-binding molecule does not contain an amino acid sequence corresponding to the amino acid sequence that crosslinks the alpha chain and the beta chain of native proHp.

[0107] In certain embodiments, the N-terminally truncated haptoglobin alpha chain of the recombinant hemoglobin-binding molecule is joined to a haptoglobin beta chain or hemoglobin-binding fragment thereof described herein by a disulfide bond formed between a cysteine ​​residue in the haptoglobin beta chain or hemoglobin-binding fragment thereof and a cysteine ​​residue among at least 14 consecutive C-terminal amino acid residues of the haptoglobin alpha chain. In certain embodiments, the haptoglobin beta chain or hemoglobin-binding fragment thereof comprises an amino acid sequence having at least 80% sequence identity with amino acid residues 162-406 of SEQ ID NO:1.

[0108] In another embodiment, the hemoglobin-binding molecule further comprises a further functional moiety. In an embodiment, the further functional moiety is conjugated to the N-terminal truncated haptoglobin alpha chain. Those skilled in the art are familiar with suitable functional moieties, and illustrative examples thereof are described elsewhere herein. In an embodiment, the further functional moiety is selected from the group consisting of a heme-binding moiety, an immunoglobulin Fc domain or its FcRn-binding fragment, and albumin. In an embodiment, the further functional moiety is a heme-binding moiety. In a preferred embodiment, the heme-binding moiety is hemopexin or its heme-binding fragment.

[0109] In one embodiment, the composition comprises about 2 μM to about 20 mM of a recombinant haptoglobin beta chain or a hemoglobin-binding fragment thereof. In one embodiment, the composition comprises about 2 μM to about 5 mM of a recombinant haptoglobin beta chain or a hemoglobin-binding fragment thereof. In one embodiment, the composition comprises about 100 μM to about 5 mM of a recombinant haptoglobin beta chain or a hemoglobin-binding fragment thereof, or a functional analog thereof. In one embodiment, the composition comprises about 2 μM to about 300 μM of a recombinant haptoglobin beta chain or a hemoglobin-binding fragment thereof. In one embodiment, the composition comprises about 5 μM to about 50 μM of a recombinant haptoglobin beta chain or a hemoglobin-binding fragment thereof. In one embodiment, the composition comprises about 10 μM to about 30 μM of a recombinant haptoglobin beta chain or a hemoglobin-binding fragment thereof.

[0110] The pharmaceutical compositions disclosed herein may be formulated for any suitable route of administration, illustrative examples of which include intravascular, intrathecal, intracranial, and intraventricular administration.

[0111] In an embodiment, the pharmaceutical composition disclosed herein is formulated for intrathecal administration.Those skilled in the art are familiar with suitable intrathecal delivery systems, and illustrative examples thereof are described by Kilburn et al. (2013, "Intrathecal Administration", Rudek M., Chau C., Figg W., McLeod H. (eds.), "Handbook of Anticancer Pharmacokinetics and Pharmacodynamics.Cancer Drug Discovery and Development", Springer, New York, NY), the contents of which are incorporated herein by reference in their entirety.

[0112] In another embodiment, the pharmaceutical compositions disclosed herein are formulated for intracranial administration.Those skilled in the art are familiar with suitable intracranial delivery systems, an illustrative example of which is described by Upadhyay et al. (2014, PNAS, 111(45):16071-16076), the contents of which are incorporated herein by reference in their entirety.

[0113] In another embodiment, the pharmaceutical compositions disclosed herein are formulated for intraventricular administration.Those skilled in the art are familiar with suitable intraventricular delivery systems, an illustrative example of which is described by Cook et al. (2009, Pharmacotherapy. 29(7):832-845), the contents of which are incorporated herein by reference in their entirety.

[0114] The disclosure also extends to unit dosage forms of the pharmaceutical compositions described herein. Suitable pharmaceutical compositions and unit dosage forms thereof can contain conventional ingredients in conventional proportions, with or without additional active compounds or major ingredients, and such unit dosage forms can contain any suitable effective amount of the active ingredient, consistent with the daily dosage range intended to be utilized.

[0115] The recombinant haptoglobin beta chain or hemoglobin-binding fragment thereof described herein, and pharmaceutical compositions comprising same, are used for the treatment of conditions associated with acellular hemoglobin (Hb), including, but not limited to, hemorrhagic stroke, sickle cell disease, red blood cell lysis, and hemoglobinopathies.

[0116] Hemorrhagic stroke is typically characterized by the rupture of blood vessels in the brain, causing localized bleeding (haemorrhage). The location of the hemorrhage may vary, and the type of hemorrhagic stroke is characterized by this location. Examples of hemorrhagic stroke include: i) intracerebral hemorrhage, involving the rupture of a blood vessel in the brain; ii) intraventricular hemorrhage, which is bleeding into the cerebral vasculature; and iii) subarachnoid hemorrhage (SAH), involving bleeding in the space between the brain and the tissues covering it, known as the subarachnoid space. SAH is most often caused by a ruptured aneurysm, referred to as aneurysmal subarachnoid hemorrhage (aSAH). Other causes of SAH include head injury, bleeding disorders, and the use of anticoagulants. Hemorrhagic stroke consists of a range of conditions with different natural histories, evaluations, and management, as those skilled in the art are familiar with. It is generally classified according to etiology as primary hemorrhagic stroke or secondary hemorrhagic stroke.

[0117] Those skilled in the art are familiar with methods for diagnosing hemorrhagic stroke, and in particular SAH, in a subject, illustrative examples of which include cerebral angiography, computed tomography (CT), and spectrophotometric analysis of oxyHb and bilirubin in the subject's CSF (see, e.g., Cruickshank AM., 2001, "ACP Best Practice No 166", J. Clin. Path., 54(11):827-830).

[0118] Those skilled in the art will appreciate that hemorrhagic stroke can be spontaneous (e.g., as a result of a ruptured aneurysm) or traumatic (e.g., as a result of trauma to the head). In some embodiments, hemorrhagic stroke is spontaneous, also known as non-traumatic hemorrhage. In some embodiments, hemorrhagic stroke is traumatic.

[0119] In some embodiments, the hemorrhagic stroke is an intraventricular hemorrhage or a subarachnoid hemorrhage. The subarachnoid hemorrhage can be an aneurysmal subarachnoid hemorrhage (aSAH).

[0120] Hemoglobinopathies are a group of genetic disorders characterized by genetic abnormalities that affect hemoglobin. These abnormalities are caused by mutations and / or deletions in the alpha globin gene or in the beta globin gene. Examples of hemoglobinopathies include sickle cell disease, which is associated with structural abnormalities in hemoglobin, and thalassemia, which is associated with defective hemoglobin production. Other hemoglobinopathies associated with red blood cell lysis and release of cell-free Hb are also known to those skilled in the art. There are many forms of thalassemia, broadly classified as alpha thalassemia and beta thalassemia, depending on whether they are associated with defective alpha or beta globin chain synthesis. Each hemoglobinopathic disorder is associated with a unique and highly variable pathology that differs in natural history, evaluation, and management, as those skilled in the art will be familiar with. Those skilled in the art will be familiar with how to diagnose hemoglobinopathies in a subject. For example, diagnosis may involve a red blood cell count with red blood cell indices and hemoglobin tests such as electrophoresis and / or chromatography for hemoglobin followed by DNA testing if applicable.

[0121] In some embodiments, the hemoglobinopathy is sickle cell disease. In other embodiments, the hemoglobinopathy is thalassemia. The thalassemia can be α-thalassemia or β-thalassemia.

[0122] Those skilled in the art will understand that the recombinant haptoglobin beta chain or hemoglobin-binding fragment thereof, or pharmaceutical composition disclosed herein is suitable for use in the treatment or prevention of any disease, condition, or disorder associated with acellular Hb. Such diseases, conditions, and disorders are known to those skilled in the art.

[0123] As used herein, the term "therapeutically effective amount" means that the amount or concentration of recombinant haptoglobin beta chain or hemoglobin-binding fragment thereof is sufficient to allow Hp to bind to and form a complex with the cell-free Hb present, thereby neutralizing the otherwise harmful biological effects of cell-free Hb. It will be understood by those skilled in the art that the therapeutically effective amount of a peptide may vary depending on several factors, illustrative examples of which include whether the recombinant haptoglobin beta chain or hemoglobin-binding fragment thereof is administered directly to the subject (e.g., intravascularly, intrathecally, intracranially, or intraventricularly) or as a pharmaceutical composition, the health and general condition of the subject being treated, the taxonomic group of the subject being treated, the severity of the condition (e.g., the extent of bleeding), the route of administration, the concentration and / or amount of cell-free Hb being neutralized, and any combination of the foregoing.

[0124] As used herein, the terms "treating", "treatment", "treating", and the like are used interchangeably to mean alleviating, minimizing, alleviating, ameliorating, or otherwise inhibiting one or more symptoms associated with a condition associated with acellular Hb. As used herein, the terms "treating", "treatment", and the like are also used interchangeably to mean preventing the occurrence of a condition associated with acellular Hb, or delaying the onset or subsequent progression of a condition associated with acellular Hb in a subject who is predisposed to or at risk of developing a condition associated with acellular Hb, but has not yet been diagnosed as having it. In this context, the terms "treating", "treatment", and the like are used interchangeably with the terms "prevention", "prophylactic", and "preventative". However, it should be understood that the methods disclosed herein do not necessarily completely prevent a condition associated with acellular Hb from occurring in the subject being treated. The methods disclosed herein may be sufficient to alleviate, reduce, alleviate, ameliorate, or otherwise inhibit conditions associated with cell-free Hb in a subject to an extent that there are fewer symptoms and / or less severe adverse symptoms than they would otherwise be observed in the absence of treatment. Thus, the methods described herein may reduce the number and / or severity of conditions associated with cell-free Hb.

[0125] The therapeutically effective amount of recombinant haptoglobin beta chain or hemoglobin-binding fragment thereof will typically fall within a relatively broad range as determined by one of skill in the art. Illustrative examples of suitable therapeutically effective amounts of recombinant haptoglobin beta chain or hemoglobin-binding fragment thereof include about 2 μM to about 20 mM, preferably about 2 μM to about 5 mM, preferably about 100 μM to about 5 mM, preferably about 2 μM to about 300 μM, preferably about 5 μM to about 100 μM, preferably about 5 μM to about 50 μM, or more preferably about 10 μM to about 30 μM.

[0126] In one embodiment, the therapeutically effective amount of the recombinant haptoglobin beta chain or hemoglobin-binding fragment thereof is about 2 μM to about 20 mM. In one embodiment, the therapeutically effective amount of the recombinant haptoglobin beta chain or hemoglobin-binding fragment thereof is about 2 μM to about 5 mM. In one embodiment, the therapeutically effective amount of the recombinant haptoglobin beta chain or hemoglobin-binding fragment thereof is about 100 μM to about 5 mM. In one embodiment, the therapeutically effective amount of the recombinant haptoglobin beta chain or hemoglobin-binding fragment thereof is about 2 μM to about 300 μM. In one embodiment, the therapeutically effective amount of the recombinant haptoglobin beta chain or hemoglobin-binding fragment thereof is about 5 μM to about 50 μM. In one embodiment, the therapeutically effective amount of the recombinant haptoglobin beta chain or hemoglobin-binding fragment thereof is about 10 μM to about 30 μM.

[0127] In an embodiment, the therapeutically effective amount of the recombinant haptoglobin beta chain or hemoglobin-binding fragment thereof is at least equimolar to the concentration of cell-free Hb to be neutralized. In the case of hemorrhagic stroke, the therapeutically effective amount of the recombinant haptoglobin beta chain or hemoglobin-binding fragment thereof is an amount sufficient to complex about 3 μM to about 300 μM of cell-free Hb in the CSF. Suitable methods for measuring the concentration of cell-free Hb in the CSF are known to those skilled in the art, and illustrative examples thereof are described in Cruickshank AM., 2001, "ACP Best Practice No 166", J.Clin.Path., 54(11):827-830; and Hugelshofer M. et al., 2018, World Neurosurg., 120:e660-e666, the contents of which are incorporated herein by reference in their entirety.

[0128] The dosage of recombinant haptoglobin beta chain or hemoglobin-binding fragment thereof is also adjusted to provide the optimal therapeutic response, for example, several divided doses are administered daily, weekly or at other appropriate time intervals, or the dosage is reduced as indicated by the demands of the situation.

[0129] In one embodiment, the administered amount of recombinant haptoglobin beta chain or hemoglobin-binding fragment thereof is sufficient to substantially neutralize cell-free Hb. "Substantially neutralize" refers to a biological effect of at least 10%, preferably about 10% to about 20%, preferably about 15% to about 25%, preferably about 20% to about 30%, preferably about 25% to about 35%, preferably about 30% to about 40%, compared to the biological effect of cell-free Hb in the absence of a therapeutic recombinant haptoglobin beta chain or hemoglobin-binding fragment thereof as described herein. Preferably, about 35% to about 45%, preferably about 40% to about 50%, preferably about 45% to about 55%, preferably about 50% to about 60%, preferably about 55% to about 65%, preferably about 60% to about 70%, preferably about 65% to about 75%, preferably about 70% to about 80%, preferably about 75% to about 85%, preferably about 80% to about 90%, preferably about 85% to about 95%, or , 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 120, 121, 122, 123, 124, 125, 126, 127, 128, 130, 132, 133, 134, 135, 136, 137, 138, 140, 141, 142, 143, 144, 145, 146, 147, 148, 150, 152, 154, 156, 158, 160, 161, 162, 163, 164, 165, 166, 167, 170, 172, 174 , 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% reduction. One of skill in the art will be familiar with how the amount of cell-free Hb may be measured or determined (either qualitatively or quantitatively).

[0130] The present invention also provides a method for treating or preventing a condition associated with cell-free hemoglobin (Hb) in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a recombinant haptoglobin beta chain or a hemoglobin-binding fragment thereof, prepared according to the methods described herein, for a period of time sufficient to allow the haptoglobin beta chain or a hemoglobin-binding fragment thereof to form a complex with, and thereby neutralize, the cell-free Hb.

[0131] The haptoglobin beta chain or its hemoglobin-binding fragment and pharmaceutical composition described herein can be administered to a subject by any suitable method known in the art.For example, the haptoglobin beta chain or its hemoglobin-binding fragment and pharmaceutical composition described herein can be administered by oral delivery, injection, parenteral delivery, subcutaneous delivery, intravenous delivery, intravitreal delivery, or intramuscular delivery.In some embodiments, the haptoglobin beta chain or its hemoglobin-binding fragment and pharmaceutical composition can also be formulated for sustained delivery.

[0132] In certain embodiments, the method comprises intravascularly administering to the subject a therapeutically effective amount of a recombinant haptoglobin beta chain, or a hemoglobin-binding fragment thereof.

[0133] In certain embodiments, the method comprises intracranially administering to the subject a therapeutically effective amount of a recombinant haptoglobin beta chain, or a hemoglobin-binding fragment thereof.

[0134] In some embodiments, the method comprises administering intrathecally to a subject a therapeutically effective amount of a recombinant haptoglobin beta chain or a hemoglobin-binding fragment thereof. In some embodiments, the method comprises administering intrathecally to the spinal canal of a subject a therapeutically effective amount of a recombinant haptoglobin beta chain or a hemoglobin-binding fragment thereof. In some embodiments, the method comprises administering intrathecally to the subarachnoid space of a subject a therapeutically effective amount of a recombinant haptoglobin beta chain or a hemoglobin-binding fragment thereof.

[0135] In certain embodiments, the method comprises administering intracerebroventricularly to the subject a therapeutically effective amount of a recombinant haptoglobin beta chain, or a hemoglobin-binding fragment thereof.

[0136] As used herein, the term "subject" refers to a mammalian subject for whom treatment or prevention is desired. Illustrative examples of suitable subjects include primates, particularly humans, pet animals such as cats and dogs, working animals such as horses, donkeys, livestock such as sheep, cows, goats, pigs, laboratory animals such as rabbits, mice, rats, guinea pigs, hamsters, and captive wild animals such as deer, dingoes, and captive wild animals in zoos and wildlife parks. In an embodiment, the subject is a human. In a further embodiment, the subject is a pediatric patient aged (i) from birth to about 2 years of age, (ii) from about 2 to about 12 years of age, or (iii) from about 12 to about 21 years of age.

[0137] The present disclosure also extends to the use of a therapeutically effective amount of a recombinant haptoglobin beta chain, or a hemoglobin-binding fragment thereof, prepared according to the methods described herein, in the manufacture of a medicament for the treatment or prevention of a condition associated with acellular hemoglobin (Hb) in a subject.

[0138] auxiliary treatment The methods of treating or preventing a condition associated with acellular Hb described herein may be suitable to be performed sequentially or in combination (e.g., simultaneously) with one or more additional treatment strategies designed to reduce, inhibit, prevent, or otherwise alleviate a condition associated with acellular Hb. In certain embodiments, the methods described herein further comprise administering to the subject at least one additional therapeutic agent for treating or preventing a condition associated with red blood cell lysis and release of acellular Hb. Those skilled in the art will be familiar with suitable adjunctive therapies and therapeutic agents for treating or preventing one or more conditions associated with acellular hemoglobin (Hb), illustrative examples of which include: (i) Correction of coagulopathy: for example, using vitamin K antagonists (VKAs), novel oral anticoagulants (NOACs such as dabigatran, rivaroxaban, and apixaban), FEIBA (Factor VIII inhibitor bypass activator), and activated recombinant factor VII (rFVIIa), prothrombin complex concentrates, activated charcoal, antiplatelet therapy (APT), and aspirin monotherapy; (ii) Antihypertensive: for example, illustrative examples of which are (i) thiazides, including chlorthalidone, chlorthiazide, dichlorophenamide, hydroflumethiazide, indapamide, and hydrochlorothiazide; loop diuretics, such as bumetanide, ethacrynic acid, furosemide, and torsemide; potassium sparing agents, such as amiloride and triamterene; and aldosterone antagonists, such as spironolactone, epirenone; (ii) diuretics, such as acebutolol, atenolol, betaxolol, (iii) beta-blockers such as bevantolol, bisoprolol, bopindolol, carteolol, carvedilol, celiprolol, esmolol, indenolol, metaprolol, nadolol, nebivolol, penbutolol, pindolol, propanolol, sotalol, tertatolol, tilisolol, and timolol; (iv) amlodipine, aranidipine, azelnidipine, barnidipine, benidipine, bepridil, cinardipine, clevidipine, diltiazem, efonidipine, felodipine, Calcium channel blockers such as acetaminophen, gallopamil, isradipine, lacidipine, remildipine, lercanidipine, nicardipine, nifedipine, nilvadipine, nimodepine, nisoldipine, nitrendipine, manidipine, pranidipine, and verapamil; (iv) benazepril; captopril; cilazapril; delapril; enalapril; fosinopril; imidapril; rosinopril; moexipril; quinapril; quinaprilat; ramipril; perindopril; perindropril; quanipril; (v) neutral endopeptidase inhibitors such as omapatrilat, cadoxatril and ecadotril, fosidotril, sampatrilat, AVE7688, ER4030; (vi) endothelin antagonists such as tezosentan, A308165, and YM62899; (vii) vasodilators such as hydralazine, clonidine, minoxidil, and nicotinyl alcohol;(viii) angiotensin II receptor antagonists such as candesartan, eprosartan, irbesartan, losartan, pratosartan, tasosartan, telmisartan, valsartan, and EXP-3137, FI6828K, and RNH6270; (ix) alpha / beta blockers such as nipradilol, arotinolol, and amosulalol; (x) alpha 1 blockers such as terazosin, urapidil, prazosin, bunazosin, trimazosin, doxazosin, naftopidil, indoramin, WHIP164, and XENOlO; and (xi) antihypertensives including alpha 2 agonists such as lofexidine, tiamenidine, moxonidine, rilmenidine, and guanobenz; (ii-b) Vasodilators: for example, hydralazine (apresoline), clonidine (catapres), minoxidil (loniten), nicotinyl alcohol (loniacol), sydnon, and sodium nitroprusside; (iii) seizure, glucose, and temperature management: e.g., antiepileptic drugs, insulin infusions to control blood glucose levels, maintenance of normothermia, and therapeutic hypothermia; (iv) Surgical treatment: for example, hematoma evacuation (surgical clot removal), decompressive craniectomy (DC), minimally invasive surgery (MIS; e.g., needle aspiration of basal ganglia hemorrhage), MIS with recombinant tissue-type plasminogen activator (rtPA); (v) timing of surgery: for example, 4–96 hours after onset; (vi) Thrombin inhibitors: e.g., hirudin, argatroban, serine protease inhibitors (e.g., nafamostat mesylate); (vii) Prevention of heme and iron toxicity: for example, nonspecific heme oxygenase (HO) inhibitors such as tin-mesoporphyrin, iron chelators such as deferoxamine; (viii) PPARg antagonists and agonists: for example, rosiglitazone, 15d-PGJ2, and pioglitazone; (ix) inhibition of microglial activation: for example, tuftusin fragment 1-3 (microglia / macrophage inhibitory factor) or minocycline (an antibiotic of the tetracycline class); (x) Upregulation of Nrf2 (NF-erythroid-2-related factor 2); (xi) Inhibition of cyclooxygenase (COX): for example, celecoxib (selective COX-2 inhibitor); (xii) matrix metalloproteinase; (xiii) TNF-α modulators: for example, adenosine receptor agonists such as CGS21680, TNF-α-specific antisense oligodeoxynucleotides such as ORF4-PE; (xiv) hypertensives: e.g., catecholamines; and (xv) Inhibitors of TLR4 signaling: for example, antibodies, Mts510 and TAK-242 (cyclohexane derivatives) Includes.

[0139] In an embodiment, the additional therapeutic agent is one or more functional moieties to which the N-terminal truncated proHp is linked, conjugated, tethered or otherwise attached, as described elsewhere herein. In an embodiment, the additional therapeutic agent is selected from the group consisting of immunoglobulin Fc region or Fc receptor binding fragments thereof, albumin or fragments thereof, hemopexin, transferrin or fragments thereof, C-terminal peptide of human chorionic gonadotropin, XTEN sequence, homoamino acid repeat (HAP), proline-alanine-serine repeat (PAS), afamin, alpha-fetoprotein, vitamin D binding protein, polypeptides capable of binding to albumin or immunoglobulin constant region under physiological conditions, neonatal Fc receptor (FcRn), in particular, immunoglobulin constant region and parts thereof, preferably polypeptides capable of binding to the Fc portion of immunoglobulin, and any combination of the above. In another embodiment, the functional moiety is selected from the group consisting of hydroxyethyl starch (HES), polyethylene glycol (PEG), polysialic acid (PSA), elastin-like polypeptides, heparosan polymers, hyaluronic acid and albumin binding ligands, e.g., fatty acid chains, and any combination of the foregoing.

[0140] In some embodiments, the additional therapeutic agent is a vasodilator. Those skilled in the art are familiar with suitable vasodilators, illustrative examples of which include sydnone and sodium nitroprusside. Thus, in some embodiments disclosed herein, the additional therapeutic agent is selected from the group consisting of sydnone and sodium nitroprusside.

[0141] Suitable adjunctive therapies for the treatment of hemoglobinopathies, such as sickle cell disease and α- or β-thalassemia, include bone marrow transplantation and / or blood transfusions. Additional therapeutic agents used to treat the symptoms of sickle cell disease may include analgesics, antibiotics, ACE inhibitors, hydroxyurea, L-glutamine, iron chelators, folic acid, hemoglobin oxygen affinity modulators (e.g., voxelotor), and antibodies (e.g., crizanluzumab).

[0142] Those skilled in the art will be aware that the invention described herein is subject to variations and modifications other than those specifically described.It is to be understood that the invention described herein includes all such variations and modifications.The invention also includes all such steps, compositions, methods, compositions, and compounds, individually or collectively referred to or indicated herein, and any and all combinations of any two or more of the steps or compositions described above.

[0143] Certain embodiments of the invention will now be described with reference to the following examples, which are intended for purposes of illustration only and are not intended to limit the scope of the generality described hereinbefore.

[0144] Sequence Listing: SEQ ID NO: 1: proHp, precursor of haptoglobin 2FS human Hp isoform 1; NP_005134 1 MSALGAVIAL LLWGQLFAVD SGNDVTDIAD DGCPKPPEIA HGYVEHSVRY QCKNYYKLRT 61 EGDGVYTLND KKQWINKAVG DKLPECEADD GCPKPPEIAH GYVEHSVRYQ CKNYYKLRTE 121 GDGVYTLNNE KQWINKAVGD KLPECEAVCG KPKNPANPVQ RILGGHLDAK GSFPWQAKMV 181 SHHNLTTGAT LINEQWLLTT AKNLFLNHSE NATAKDIAPT LTLYVGKKQL VEIEKVVLHP 241 NYSQVDIGLI KLKQKVSVNE RVMPICLPSK DYAEVGRVGY VSGWGRNANF KFTDHLKYVM 301 LPVADQDQCI RHYEGSTVPE KKTPKSPVGV QPILNEHTFC AGMSKYQEDT CYGDAGSAFA 361 VHDLEEDTWY ATGILSFDKS CAVAEYGVYV KVTSIQDWVQ KTIAEN SEQ ID NO:2: proHp, the precursor of human Hp isoform 2; NP_001119574 1 MSALGAVIAL LLWGQLFAVD SGNDVTDIAD DGCPKPPEIA HGYVEHSVRY QCKNYYKLRT 61 EGDGVYTLNN EKQWINKAVG DKLPECEAVC GKPKNPANPV QRILGGHLDA KGSFPWQAKM 121 VSHHNLTTGA TLINEQWLLT TAKNLFLNHS ENATAKDIAP TLTLYVGKKQ LVEIEKVVLH 181 PNYSQVDIGL IKLKQKVSVN ERVMPICLPS KDYAEVGRVG YVSGWGRNAN FKFTDHLKYV 241 MLPVADQDQC IRHYEGSTVP EKKTPKSPVG VQPILNEHTF CAGMSKYQED TCYGDAGSAF 301 AVHDLEEDTW YATGILSFDK SCAVAEYGVY VKVTSIQDWV QKTIAEN SEQ ID NO: 3: proHp, the precursor of human Hp isoform 3; NP_001305067 1 MSALGAVIAL LLWGQLFAVD SGNDVTDIAD DGCPKPPEIA HGYVEHSVRY QCKNYYKLRT 61 EGDGVYTLND KKQWINKAVG DKLPECEAVC GKPKNPANPV QRILGGHLDA KGSFPWQAKM 121 VSHHNLTTGA TLINEQWLLT TAKNLFLNHS ENATAKDIAP TLTLYVGKKQ LVEIEKVVLH 181 PNYSQVDIGL IKLKQKVSVN ERVMPICLPS KDYAEVGRVG YVSGWGRNAN FKFTDHLKYV 241 MLPVADQDQC IRHYEGSTVP EKKTPKSPVG VQPILNEHTF CAGMSKYQED TCYGDAGSAF 301 AVHDLEEDTW YATGILSFDK SCAVAEYGVY VKVTSIQDWV QKTIAEN SEQ ID NO: 4: Human C1r-LP; NP_057630 1 MPGPRVWGKY LWRSPHSKGC PGAMWWLLLW GVLQACPTRG SVLLAQELPQ QLTSPGYPEP 61 YGKGQESSTD IKAPEGFAVR LVFQDFDLEP SQDCAGDSVT ISFVGSDPSQ FCGQQGSPLG 121 RPPGQREFVS SGRSLRLTFR TQPSSENKTA HLHKGFLALY QTVAVNYSQP ISEASRGSEA 181 INAPGDNPAK VQNHCQEPYY QAAAAGALTC ATPGTWKDRQ DGEEVLQCMP VCGRPVTPIA 241 QNQTTLGSSR AKLGNFPWQA FTSIHGRGGG ALLGDRWILT AAHTIYPKDS VSLRKNQSVN 301 VFLGHTAIDE MLKLGNHPVH RVVVHPDYRQ NESHNFSGDI ALLELQHSIP LGPNVLPVCL 361 PDNETLYRSG LLGYVSGFGM EMGWLTTELK YSRLPVAPRE ACNAWLQKRQ RPEVFSDNMF 421 CVGDETQRHS VCQGDSGSVY VVWDNHAHHW VATGIVSWGI GCGEGYDFYT KVLSYVDWIK 481 GVMNGKN SEQ ID NO: 5: Human C1r-LP; NP_001284569 1 MPGPRVWGKY LWRSPHSKGC PGAMWWLLLW GVLQACPTRG SVLLAQELPQ QLTSPGYPEP 61 YGKGQESSTD IKAPEGFAVR LVFQDFDLEP SQDCAGDSVT ISFVGSDPSQ FCGQQGSPLG 121 RPPGQREFVS SGRSLRLTFR TQPSSENKTA HLHKGFLALY QTVGALTCAT PGTWKDRQDG 181 EEVLQCMPVC GRPVTPIAQN QTTLGSSRAK LGNFPWQAFT SIHGRGGGAL LGDRWILTAA 241 HTIYPKDSVS LRKNQSVNVF LGHTAIDEML KLGNHPVHRV VVHPDYRQNE SHNFSGDIAL 301 LELQHSIPLG PNVLPVCLPD NETLYRSGLL GYVSGFGMEM GWLTTELKYS RLPVAPREAC 361 NAWLQKRQRP EVFSDNMFCV GDETQRHSVC QGDSGSVYVV WDNHAHHWVA TGIVSWGIGC 421 GEGYDFYTKV LSYVDWIKGV MNGKN SEQ ID NO: 6: Human C1r-LP; NP_001284571 1 MPGPRVWGKY LWRSPHSKGC PGAMWWLLLW GVLQACPTRG SVLLAQELPQ QLTSPGYPEP 61 YGKGQESSTD IKAPEGFAVR LVFQDFDLEP SQDCAGDSVT ISFVGSDPSQ FCGQQGSPLG 121 RPPGQREFVS SGRSLRLTFR TQPSSENKTA HLHKGFLALY QTVAVNYSQP ISEASRGSEA 181 INAPGDNPAK VQNHCQEPYY QAAAAASTPS LFLCLSSFTP QGHSPVQPQG PGKTDRMGRR 241 FFSVCLSADG QSPPLPRIRR PSVLPEPSWA TSPGKPSPVS TAVGAGPCWG TDGSSLLPTP 301 STPRTVFLSG RTRV SEQ ID NO: 7: Human C1r-LP; NP_001284572 1 MPGPRVWGKY LWRSPHSKGC PGAMWWLLLW GVLQACPTRG SVLLAQELPQ QLTSPGYPEP 61 YGKGQESSTD IKAPEGFAVR LVFQDFDLEP SQDCAGDSVT ISFVGSDPSQ FCGQQGSPLG 121 RPPGQREFVS SGRSLRLTFR TQPSSENKTA HLHKGFLALY QTVGECPSWG CREGASVPSH 181 DPGIFKP SEQ ID NO: 8: 14 consecutive C-terminal amino acid residues of the Hp alpha chain VCGKPKNPANPVQR SEQ ID NO: 9: Human serum albumin (HAS); NP_000468 1 MKWVTFISLL FLFSSAYSRG VFRRDAHKSE VAHRFKDLGE ENFKALVLIA FAQYLQQCPF 61 EDHVKLVNEV TEFAKTCVAD ESAENCDKSL HTLFGDKLCT VATLRETYGE MADCCAKQEP 121 ERNECFLQHK DDNPNLPRLV RPEVDVMCTA FHDNEETFLK KYLYEIARRH PYFYAPELLF 181 FAKRYKAAFT ECCQAADKAA CLLPKLDELR DEGKASSAKQ RLKCASLQKF GERAFKAWAV 241 ARLSQRFPKA EFAEVSKLVT DLTKVHTECC HGDLLECADD RADLAKYICE NQDSISSKLK 301 ECCEKPLLEK SHCIAEVEND EMPADLPSLA ADFVESKDVC KNYAEAKDVF LGMFLYEYAR 361 RHPDYSVVLL LRLAKTYETT LEKCCAAADP HECYAKVFDE FKPLVEEPQN LIKQNCELFE 421 QLGEYKFQNA LLVRYTKKVP QVSTPTLVEV SRNLGKVGSK CCKHPEAKRM PCAEDYLSVV 481 LNQLCVLHEK TPVSDRVTKC CTESLVNRRP CFSALEVDET YVPKEFNAET FTFHADICTL 541 SEKERQIKKQ TALVELVKHK PKATKEQLKA VMDDFAAFVE KCCKADDKET CFAEEGKKLV 601 AASQAALGL SEQ ID NO: 10: Human CD163; NP_981961 1 MSKLRMVLLE DSGSADFRRH FVNLSPFTIT VVLLLSACFV TSSLGGTDKE LRLVDGENKC 61 SGRVEVKVQE EWGTVCNNGW SMEAVSVICN QLGCPTAIKA PGWANSSAGS GRIWMDHVSC 121 RGNESALWDC KHDGWGKHSN CTHQQDAGVT CSDGSNLEMR LTRGGNMCSG RIEIKFQGRW 181 GTVCDDNFNI DHASVICRQL ECGSAVSFSG SSNFGEGSGP IWFDDLICNG NESALWNCKH 241 QGWGKHNCDH AEDAGVICSK GADLSLRLVD GVTECSGRLE VRFQGEWGTI CDDGWDSYDA 301 AVACKQLGCP TAVTAIGRVN ASKGFGHIWL DSVSCQGHEP AIWQCKHHEW GKHYCNHNED 361 AGVTCSDGSD LELRLRGGGS RCAGTVEVEI QRLLGKVCDR GWGLKEADVV CRQLGCGSAL 421 KTSYQVYSKI QATNTWLFLS SCNGNETSLW DCKNWQWGGL TCDHYEEAKI TCSAHREPRL 481 VGGDIPCSGR VEVKHGDTWG SICDSDFSLE AASVLCRELQ CGTVVSILGG AHFGEGNGQI 541 WAEEFQCEGH ESHLSLCPVA PRPEGTCSHS RDVGVVCSRY TEIRLVNGKT PCEGRVELKT 601 LGAWGSLCNS HWDIEDAHVL CQQLKCGVAL STPGGARFGK GNGQIWRHMF HCTGTEQHMG 661 DCPVTALGAS LCPSEQVASV ICSGNQSQTL SSCNSSSLGP TRPTIPEESA VACIESGQLR 721 LVNGGGRCAG RVEIYHEGSW GTICDDSWDL SDAHVVCRQL GCGEAINATG SAHFGEGTGP 781 IWLDEMKCNG KESRIWQCHS HGWGQQNCRH KEDAGVICSE FMSLRLTSEA SREACAGRLE 841 VFYNGAWGTV GKSSMSETTV GVVCRQLGCA DKGKINPASL DKAMSIPMWV DNVQCPKGPD 901 TLWQCPSSPW EKRLASPSEE TWITCDNKIR LQEGPTSCSG RVEIWHGGSW GTVCDDSWDL 961 DDAQVVCQQL GCGPALKAFK EAEFGQGTGP IWLNEVKCKG NESSLWDCPA RRWGHSECGH 1021 KEDAAVNCTD ISVQKTPQKA TTGRSSRQSS FIAVGILGVV LLAIFVALFF LTKKRRQRQR 1081 LAVSSRGENL VHQIQYREMN SCLNADDLDL MNSSGGHSEP H SEQ ID NO: 11: Human LRP1; NP_002323 1 MLTPPLLLLL PLLSALVAAA IDAPKTCSPK QFACRDQITC ISKGWRCDGE RDCPDGSDEA 61 PEICPQSKAQ RCQPNEHNCL GTELCVPMSR LCNGVQDCMD GSDEGPHCRE LQGNCSRLGC 121 QHHCVPTLDG PTCYCNSSFQ LQADGKTCKD FDECSVYGTC SQLCTNTDGS FICGCVEGYL 181 LQPDNRSCKA KNEPVDRPPV LLIANSQNIL ATYLSGAQVS TITPTSTRQT TAMDFSYANE 241 TVCWVHVGDS AAQTQLKCAR MPGLKGFVDE HTINISLSLH HVEQMAIDWL TGNFYFVDDI 301 DDRIFVCNRN GDTCVTLLDL ELYNPKGIAL DPAMGKVFFT DYGQIPKVER CDMDGQNRTK 361 LVDSKIVFPH GITLDLVSRL VYWADAYLDY IEVVDYEGKG RQTIIQGILI EHLYGLTVFE 421 NYLYATNSDN ANAQQKTSVI RVNRFNSTEY QVVTRVDKGG ALHIYHQRRQ PRVRSHACEN 481 DQYGKPGGCS DICLLANSHK ARTCRCRSGF SLGSDGKSCK KPEHELFLVY GKGRPGIIRG 541 MDMGAKVPDE HMIPIENLMN PRALDFHAET GFIYFADTTS YLIGRQKIDG TERETILKDG 601 IHNVEGVAVD WMGDNLYWTD DGPKKTISVA RLEKAAQTRK TLIEGKMTHP RAIVVDPLNG 661 WMYWTDWEED PKDSRRGRLE RAWMDGSHRD IFVTSKTVLW PNGLSLDIPA GRLYWVDAFY 721 DRIETILLNG TDRKIVYEGP ELNHAFGLCH HGNYLFWTEY RSGSVYRLER GVGGAPPTVT 781 LLRSERPPIF EIRMYDAQQQ QVGTNKCRVN NGGCSSLCLA TPGSRQCACA EDQVLDADGV 841 TCLANPSYVP PPQCQPGEFA CANSRCIQER WKCDGDNDCL DNSDEAPALC HQHTCPSDRF 901 KCENNRCIPN RWLCDGDNDC GNSEDESNAT CSARTCPPNQ FSCASGRCIP ISWTCDLDDD 961 CGDRSDESAS CAYPTCFPLT QFTCNNGRCI NINWRCDNDN DCGDNSDEAG CSHSCSSTQF 1021 KCNSGRCIPE HWTCDGDNDC GDYSDETHAN CTNQATRPPG GCHTDEFQCR LDGLCIPLRW 1081 RCDGDTDCMD SSDEKSCEGV THVCDPSVKF GCKDSARCIS KAWVCDGDND CEDNSDEENC 1141 ESLACRPPSH PCANNTSVCL PPDKLCDGND DCGDGSDEGE LCDQCSLNNG GCSHNCSVAP 1201 GEGIVCSCPL GMELGPDNHT CQIQSYCAKH LKCSQKCDQN KFSVKCSCYE GWVLEPDGES 1261 CRSLDPFKPF IIFSNRHEIR RIDLHKGDYS VLVPGLRNTI ALDFHLSQSA LYWTDVVEDK 1321 IYRGKLLDNG ALTSFEVVIQ YGLATPEGLA VDWIAGNIYW VESNLDQIEV AKLDGTLRTT 1381 LLAGDIEHPR AIALDPRDGI LFWTDWDASL PRIEAASMSG AGRRTVHRET GSGGWPNGLT 1441 VDYLEKRILW IDARSDAIYS ARYDGSGHME VLRGHEFLSH PFAVTLYGGE VYWTDWRTNT 1501 LAKANKWTGH NVTVVQRTNT QPFDLQVYHP SRQPMAPNPC EANGGQGPCS HLCLINYNRT 1561 VSCACPHLMK LHKDNTTCYE FKKFLLYARQ MEIRGVDLDA PYYNYIISFT VPDIDNVTVL 1621 DYDAREQRVY WSDVRTQAIK RAFINGTGVE TVVSADLPNA HGLAVDWVSR NLFWTSYDTN 1681 KKQINVARLD GSFKNAVVQG LEQPHGLVVH PLRGKLYWTD GDNISMANMD GSNRTLLFSG 1741 QKGPVGLAID FPESKLYWIS SGNHTINRCN LDGSGLEVID AMRSQLGKAT ALAIMGDKLW 1801 WADQVSEKMG TCSKADGSGS VVLRNSTTLV MHMKVYDESI QLDHKGTNPC SVNNGDCSQL 1861 CLPTSETTRS CMCTAGYSLR SGQQACEGVG SFLLYSVHEG IRGIPLDPND KSDALVPVSG 1921 TSLAVGIDFH AENDTIYWVD MGLSTISRAK RDQTWREDVV TNGIGRVEGI AVDWIAGNIY 1981 WTDQGFDVIE VARLNGSFRY VVISQGLDKP RAITVHPEKG YLFWTEWGQY PRIERSRLDG 2041 TERVVLVNVS ISWPNGISVD YQDGKLYWCD ARTDKIERID LETGENREVV LSSNNMDMFS 2101 VSVFEDFIYW SDRTHANGSI KRGSKDNATD SVPLRTGIGV QLKDIKVFNR DRQKGTNVCA 2161 VANGGCQQLC LYRGRGQRAC ACAHGMLAED GASCREYAGY LLYSERTILK SIHLSDERNL 2221 NAPVQPFEDP EHMKNVIALA FDYRAGTSPG TPNRIFFSDI HFGNIQQIND DGSRRITIVE 2281 NVGSVEGLAY HRGWDTLYWT SYTTSTITRH TVDQTRPGAF ERETVITMSG DDHPRAFVLD 2341 ECQNLMFWTN WNEQHPSIMR AALSGANVLT LIEKDIRTPN GLAIDHRAEK LYFSDATLDK 2401 IERCEYDGSH RYVILKSEPV HPFGLAVYGE HIFWTDWVRR AVQRANKHVG SNMKLLRVDI 2461 PQQPMGIIAV ANDTNSCELS PCRINNGGCQ DLCLLTHQGH VNCSCRGGRI LQDDLTCRAV 2521 NSSCRAQDEF ECANGECINF SLTCDGVPHC KDKSDEKPSY CNSRRCKKTF RQCSNGRCVS 2581 NMLWCNGADD CGDGSDEIPC NKTACGVGEF RCRDGTCIGN SSRCNQFVDC EDASDEMNCS 2641 ATDCSSYFRL GVKGVLFQPC ERTSLCYAPS WVCDGANDCG DYSDERDCPG VKRPRCPLNY 2701 FACPSGRCIP MSWTCDKEDD CEHGEDETHC NKFCSEAQFE CQNHRCISKQ WLCDGSDDCG 2761 DGSDEAAHCE GKTCGPSSFS CPGTHVCVPE RWLCDGDKDC ADGADESIAA GCLYNSTCDD 2821 REFMCQNRQC IPKHFVCDHD RDCADGSDES PECEYPTCGP SEFRCANGRC LSSRQWECDG 2881 ENDCHDQSDE APKNPHCTSQ EHKCNASSQF LCSSGRCVAE ALLCNGQDDC GDSSDERGCH 2941 INECLSRKLS GCSQDCEDLK IGFKCRCRPG FRLKDDGRTC ADVDECSTTF PCSQRCINTH 3001 GSYKCLCVEG YAPRGGDPHS CKAVTDEEPF LIFANRYYLR KLNLDGSNYT LLKQGLNNAV 3061 ALDFDYREQM IYWTDVTTQG SMIRRMHLNG SNVQVLHRTG LSNPDGLAVD WVGGNLYWCD 3121 KGRDTIEVSK LNGAYRTVLV SSGLREPRAL VVDVQNGYLY WTDWGDHSLI GRIGMDGSSR 3181 SVIVDTKITW PNGLTLDYVT ERIYWADARE DYIEFASLDG SNRHVVLSQD IPHIFALTLF 3241 EDYVYWTDWE TKSINRAHKT TGTNKTLLIS TLHRPMDLHV FHALRQPDVP NHPCKVNNGG 3301 CSNLCLLSPG GGHKCACPTN FYLGSDGRTC VSNCTASQFV CKNDKCIPFW WKCDTEDDCG 3361 DHSDEPPDCP EFKCRPGQFQ CSTGICTNPA FICDGDNDCQ DNSDEANCDI HVCLPSQFKC 3421 TNTNRCIPGI FRCNGQDNCG DGEDERDCPE VTCAPNQFQC SITKRCIPRV WVCDRDNDCV 3481 DGSDEPANCT QMTCGVDEFR CKDSGRCIPA RWKCDGEDDC GDGSDEPKEE CDERTCEPYQ 3541 FRCKNNRCVP GRWQCDYDND CGDNSDEESC TPRPCSESEF SCANGRCIAG RWKCDGDHDC 3601 ADGSDEKDCT PRCDMDQFQC KSGHCIPLRW RCDADADCMD GSDEEACGTG VRTCPLDEFQ 3661 CNNTLCKPLA WKCDGEDDCG DNSDENPEEC ARFVCPPNRP FRCKNDRVCL WIGRQCDGTD 3721 NCGDGTDEED CEPPTAHTTH CKDKKEFLCR NQRCLSSSLR CNMFDDCGDG SDEEDCSIDP 3781 KLTSCATNAS ICGDEARCVR TEKAAYCACR SGFHTVPGQP GCQDINECLR FGTCSQLCNN 3841 TKGGHLCSCA RNFMKTHNTC KAEGSEYQVL YIADDNEIRS LFPGHPHSAY EQAFQGDESV 3901 RIDAMDVHVK AGRVYWTNWH TGTISYRSLP PAAPPTTSNR HRRQIDRGVT HLNISGLKMP 3961 RGIAIDWVAG NVYWTDSGRD VIEVAQMKGE NRKTLISGMI DEPHAIVVDP LRGTMYWSDW 4021 GNHPKIETAA MDGTLRETLV QDNIQWPTGL AVDYHNERLY WADAKLSVIG SIRLNGTDPI 4081 VAADSKRGLS HPFSIDVFED YIYGVTYINN RVFKIHKFGH SPLVNLTGGL SHASDVVLYH 4141 QHKQPEVTNP CDRKKCEWLC LLSPSGPVCT CPNGKRLDNG TCVPVPSPTP PPDAPRPGTC 4201 NLQCFNGGSC FLNARRQPKC RCQPRYTGDK CELDQCWEHC RNGGTCAASP SGMPTCRCPT 4261 GFTGPKCTQQ VCAGYCANNS TCTVNQGNQP QCRCLPGFLG DRCQYRQCSG YCENFGTCQM 4321 AADGSRQCRC TAYFEGSRCE VNKCSRCLEG ACVVNKQSGD VTCNCTDGRV APSCLTCVGH 4381 CSNGGSCTMN SKMMPECQCP PHMTGPRCEE HVFSQQQPGH IASILIPLLL LLLLVLVAGV 4441 VFWYKRRVQG AKGFQHQRMT NGAMNVEIGN PTYKMYEGGE PDDVGGLLDA DFALDPDKPT 4501 NFTNPVYATL YMGGHGSRHS LASTDEKREL LGRGPEDEIG DPLA Sequence number 12: Human hemopexin (Hpx); NP_000604 1 MARVLGAPVA LGLWSLCWSL AIATPLPPTS AHGNVAEGET KPDPDVTERC SDGWSFDATT 61 LDDNGTMLFF KGEFVWKSHK WDRELISERW KNFPSPVDAA FRQGHNSVFL IKGDKVWVYP 121 PEKKEKGYPK LLQDEFPGIP SPLDAAVECH RGECQAEGVL FFQGDREWFW DLATGTMKER 181 SWPAVGNCSS ALRWLGRYYC FQGNQFLRFD PVRGEVPPRY PRDVRDYFMP CPGRGHGHRN 241 GTGHGNSTHH GPEYMRCSPH LVLSALTSDN HGATYAFSGT HYWRLDTSRD GWHSWPIAHQ 301 WPQGPSAVDA AFSWEEKLYL VQGTQVYVFL TKGGYTLVSG YPKRLEKEVG TPHGIILDSV 361 DAAFICPGSS RLHIMAGRRL WWLDLKSGAQ ATWTELPWPH EKVDGALCME KSLGPNSCSA 421 NGPGLYLIHG PNLYCYSDVE KLNAAKALPQ PQNVTSLLGC TH SEQ ID NO: 13: Hu-LRPAP1; NP_002328 1 MAPRRVRSFL RGLPALLLLL LFLGPWPAAS HGGKYSREKN QPKPSPKRES GEEFRMEKLN 61 QLWEKAQRLH LPPVRLAELH ADLKIQERDE LAWKKLKLDG LDEDGEKEAR LIRNLNVILA 121 KYGLDGKKDA RQVTSNSLSG TQEDGLDDPR LEKLWHKAKT SGKFSGEELD KLWREFLHHK 181 EKVHEYNVLL ETLSRTEEIH ENVISPSDLS DIKGSVLHSR HTELKEKLRS INQGLDRLRR 241 VSHQGYSTEA EFEEPRVIDL WDLAQSANLT DKELEAFREE LKHFEAKIEK HNHYQKQLEI 301 AHEKLRHAES VGDGERVSRS REKHALLEGR TKELGYTVKK HLQDLSGRIS RARHNEL SEQ ID NO: 14: Amino acid sequence common to the alpha chain of Hp1 and the alpha chain of Hp2 VDSGNDVTDIADDGCPKPPEIAHGYVEHSVRYQCKNYYKLRTEGDGVYTLN SEQ ID NO: 15: Amino acid sequence of human IgG4 Fc region ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 16: Amino acid sequence of mouse IgG2a Fc region APNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISK PKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 17: Amino acid sequence common to the alpha chain of Hp1 and the alpha chain of Hp2 NEKQWINKAVGDKLPECEAVCGKPKNPANPVQR

[0145] Working Example A. Abbreviations [Table 1]

[0146] B. General Procedures B.1. Cell Culture Expi293F™ cells and mammalian expression vector, pcDNA3.1, were obtained from Invitrogen™, Thermo Fisher Scientific (R790-07, V790-20). Cells were cultured in GIBCO® Expi293 Expression Medium (Invitrogen™, Thermo Fisher Scientific). All tissue culture media was supplemented with Antibiotic-Antimycotic (GIBCO®, Thermo Fisher Scientific: 15240-096) and cells were incubated at 37°C, 8% CO 2 The cells were maintained in an incubator with an atmosphere of

[0147] B.2. Antibodies His Tag Antibody [FITC]; GenScript; Product number: A01620 Goat anti-human IgG [FITC]; Southern Biotech Polyclonal antibody against haptoglobin; Acris Antibodies; Model number: AP08546PU-N

[0148] B.3. Construction of cDNA Plasmids As used herein, the amino acid sequences of the various proteins have been recorded in the Genbank® database and have been assigned accession numbers (see Table 1 below).

[0149] [Table 2]

[0150] cDNAs, each with a Kozak consensus sequence (Kozak1987) (GCCACC) immediately upstream of the initiating methionine (at position +1), were codon-optimized and synthesized for expression in humans by Geneart® (Invitrogen™, Thermo Fisher Scientific). Standard PCR-based mutagenesis methods were used to generate mutant molecules. Once each cDNA was complete, it was digested with NheI and XhoI and ligated into pcDNA3.1 (Invitrogen™, Thermo Fisher Scientific). Large-scale preparations of plasmid DNA were performed using the QIAGEN Plasmid Giga kit (12191) according to the manufacturer's instructions. The nucleotide sequences of all plasmid constructs were verified by sequencing both strands using BigDye™ Terminator Version 3.1 Ready Reaction Cycle Sequencing (Invitrogen™, Thermo Fisher Scientific) and an Applied Biosystems 3130xl Genetic Analyzer.

[0151] B.4. Transient transfection for production of recombinant proteins Expi293F Transient transfection of expression plasmids using Expi293F cells was performed using Expifectamine™ Transfection Reagent (Invitrogen™, Life Technologies) according to the manufacturer's instructions. Cells were cultured at 1 × 10 viable cells per ml. 6 Transfect at a final concentration of 1000× and incubate at 8% CO in a shaking incubator (Infors). 2The cells were incubated at 37°C for 6 days in 5% CO. Four hours after transfection, Pluronic F68 (GIBCO, Life Technologies) was added to a final concentration of 0.1% v / v. Twenty-four hours after transfection, the cell cultures were supplemented with LucraTone Lupin (Millipore) to a final concentration of 0.5% v / v. The cell culture supernatants were harvested by centrifugation at 2500 rpm and then purified through 0.45 μm filters (Nalgene).

[0152] ExpiCHO Transient transfection of an expression plasmid encoding huLRP1 soluble mini-receptor binding domain III (90%) together with huLRPAP1 (human LDL receptor related protein associated protein 1; RAP, 10%) using ExpiCHO-S™ cells was performed using Expifectamine™ Transfection Reagent (Invitrogen, Life Technologies) according to the manufacturer's instructions. Cells were cultured at 6 x 10 viable cells per ml. 6 Transfect at a final concentration of 1000× and incubate at 8% CO in a shaking incubator (Infors). 2The cultures were incubated at 37°C for 20 hours in 5% CO2 at 32°C. After 20 hours, Enhancer™ and Feed™ were added to the cultures. The cultures were then incubated at 32°C, 5% CO2, 70% humidity for an additional 5 days. Five days after transfection, a second Feed™ was added to the cultures and they were returned to the incubator at 32°C, 5% CO2, 70% humidity. Cell culture supernatants were harvested by centrifugation at 2500 rpm and then purified by passing through a 0.45 μm filter (Nalgene). Expression of recombinant huLRP1 soluble mini-receptor in the culture supernatant was confirmed by SDS-PAGE (NuPAGE system, Thermo Fisher Scientific, MA, USA) and also by Western blot analysis using an anti-His antibody (His Tag antibody [FITC], GenScript, cat. no. A01620).

[0153] B.5. Purification of His-tagged proteins Hp(148-406) mutant The His-tagged recombinant Hp(148-406) mutant was purified on the AKTAxpress system (Cytiva) using an automated method for tandem chromatography. Specifically, 30 ml of Expi293F supernatant was diluted with 10 mM imidazole; 20 mM NaH 2 PO 4 The column was loaded onto a 1 ml HisTrap Excel column (Cytiva) equilibrated in 500 mM NaCl, pH 7.4, followed by 500 mM imidazole; 20 mM NaH 2 PO 4 Prior to elution into a retention loop using 500 mM NaCl, pH 7.4, bound His-tagged proteins were eluted with 25 mM imidazole; 20 mM NaH 2 PO 4was washed with 500 mM NaCl (pH 7.4) to reduce non-specific interacting proteins. The captured eluate from the HisTrap Excel column was then injected onto a HiPrep 26 / 10 desalting column (Cytiva) for buffer exchange into MT-PBS.

[0154] Protein-containing fractions containing all size species were pooled and concentrated using Amicon Ultra-15 centrifugal ultrafiltration devices (Merck-Millipore, MS, USA) prior to flow through a 0.22um filter. Protein concentration was then measured by OD280 using a Trinean DropSense96 system (Trinean) and purity was verified by SDS-PAGE separation on NuPAGE 4-12% Bis-Tris gels (Thermo Fisher Scientific). Higher order species levels of the solutions were assessed using an analytical Superdex 200 Increase (15 / 50) size exclusion column connected to an Agilent 1260 Infinity HPLC with MT-PBS as the mobile phase. For comparison, 1ul of Aqueous SEC1 (AL0-3042) molecular weight standard from Phenomenex was run as part of the analysis and the results were overlaid.

[0155] Hp(162-406) mutant The His-tagged recombinant Hp(162-406) mutant was purified on the AKTAxpress system (Cytiva) using an automated method for tandem chromatography. Specifically, 1-2 L of Expi293F supernatant was diluted with 10 mM imidazole; 20 mM NaH 2 PO 4 The column was loaded onto a 5 ml HisTrap Excel column (Cytiva) equilibrated in 500 mM NaCl, pH 7.4, followed by 500 mM imidazole; 20 mM NaH 2 PO 4Prior to elution into a retention loop using 500 mM NaCl, pH 7.4, bound His-tagged proteins were eluted with 25 mM imidazole; 20 mM NaH 2 PO 4 was washed with 500 mM NaCl (pH 7.4) to reduce non-specific interacting proteins. The captured eluate from the HisTrap Excel column was then injected onto a Superdex 200 26 / 60 HiPrep size exclusion column (Cytiva) for preparative separation of aggregates and size species in MT-PBS mobile phase.

[0156] Fractions containing proteins of the predicted size were pooled and concentrated using Amicon Ultra-15 centrifugal ultrafiltration devices (Merck-Millipore, MS, USA) before flow through a 0.22um filter. Protein concentration was then measured by OD280 using a Trinean DropSense96 system (Trinean) and purity was verified by SDS-PAGE separation on NuPAGE 4-12% Bis-Tris gels (Thermo Fisher Scientific). Higher order species levels of the solutions were assessed using an analytical Superdex 200 Increase (15 / 50) size exclusion column connected to an Agilent 1260 Infinity HPLC with MT-PBS as the mobile phase. For comparison, 1ul of Aqueous SEC1 (AL0-3042) molecular weight standard from Phenomenex was run as part of the analysis and the results were overlaid.

[0157] B.6. Purification of Albumin Fusion Proteins Mouse Albumin Fusion Protein Hp(148-406) mutant Mouse albumin (MSA)-fused Hp(148-406) variants were purified on the AKTAxpress system (Cytiva) using an automated method for tandem chromatography. Specifically, 30 ml of Expi293F supernatant was loaded onto a 5 ml Mimetic Blue multi species albumin affinity column (Astrea Bioseparations) equilibrated in 10 mM Tris; 150 mM NaCl, pH 7.5. The bound MSA-fusion protein was then washed with 10 mM Tris; 150 mM NaCl, pH 7.5 to reduce non-specific interacting proteins, before elution into a retention loop using 30 mM octanoate; 10 mM Tris; 150 mM NaCl, pH 7.4. The captured eluate from the Mimetic Blue column was then injected onto a HiPrep 26 / 10 desalting column (Cytiva) for buffer exchange into MT-PBS.

[0158] Protein-containing fractions containing all size species were pooled and concentrated using Amicon Ultra-15 centrifugal ultrafiltration devices (Merck-Millipore, MS, USA) prior to flow through a 0.22um filter. Protein concentration was then measured by OD280 using a Trinean DropSense96 system (Trinean) and purity was verified by SDS-PAGE separation on NuPAGE 4-12% Bis-Tris gels (Thermo Fisher Scientific). Higher order species levels of the solutions were assessed using a Superdex 200 Increase (15 / 50) size exclusion column connected to an Agilent 1260 Infinity HPLC with MT-PBS as the mobile phase. For comparison, 1ul of Aqueous SEC1 (AL0-3042) molecular weight standard from Phenomenex was run as part of the analysis and the results were overlaid.

[0159] Hp(162-406) mutant MSA-fused Hp(162-406) variants were purified on the AKTAxpress system (Cytiva) using an automated method for tandem chromatography. Specifically, 1-2 L of Expi293F supernatant was loaded onto a 5 ml Mimetic Blue multi species albumin affinity column (Astrea Bioseparations) equilibrated in 10 mM Tris; 150 mM NaCl, pH 7.5. The bound MSA-fused protein was then washed with 10 mM Tris; 150 mM NaCl, pH 7.5 to reduce non-specific interacting proteins, before elution into a retention loop using 30 mM octanoate; 10 mM Tris; 150 mM NaCl, pH 7.4. The captured eluate from the Mimetic Blue column was then injected onto a Superdex 200 26 / 60 HiPrep size exclusion column (Cytiva) for preparative separation of aggregates and size species in MT-PBS mobile phase.

[0160] Fractions containing proteins of the predicted size were pooled and concentrated using Amicon Ultra-15 centrifugal ultrafiltration devices (Merck-Millipore) prior to flow through a 0.22um filter. Protein concentration was then measured by OD280 using a Trinean DropSense96 system (Trinean) and purity was verified by SDS-PAGE separation on a NuPAGE 4-12% Bis-Tris gel (Thermo Fisher Scientific). Higher order species levels of the solutions were assessed using an analytical Superdex 200 Increase (15 / 50) size exclusion column interfaced to an Agilent 1260 Infinity HPLC with MT-PBS as the mobile phase. For comparison, 1ul of Aqueous SEC1 (AL0-3042) molecular weight standard from Phenomenex was run as part of the analysis and the results were overlaid.

[0161] Human Albumin Fusion Protein Hp(148-406) mutant Human albumin (HSA)-fused Hp(148-406) variants were purified on a Janus G3 liquid handler (Perkin Elmer) using an automated method for tandem chromatography. Specifically, 3.5 ml of Expi293F supernatant was loaded onto a 200 μl CaptureSelect HSA affinity column (Thermo) equilibrated in 10 mM Tris; 150 mM NaCl (pH 7.5). Then, 2 M MgCl 2 Prior to elution in 20 mM Tris, pH 7.4, the bound HSA fusion protein was washed with 10 mM Tris; 150 mM NaCl, pH 7.5 to reduce non-specific interacting proteins. The eluate collected from the CaptureSelect HSA affinity column was then dispensed onto a CentriPure 96 desalting array (emp Biotech GmbH) for buffer exchange into MT-PBS. The desalted sample was not further concentrated.

[0162] Protein concentration was then measured by OD280 using a Trinean DropSense96 system (Trinean) and purity was verified by SDS-PAGE separation on NuPAGE 4-12% Bis-Tris gels (Thermo Fisher Scientific). Solutions were assessed for higher molecular species levels using a Superdex 200 Increase (15 / 50) size exclusion column connected to an Agilent 1260 Infinity HPLC with MT-PBS as the mobile phase. For comparison, 1 ul of Aqueous SEC1 (AL0-3042) molecular weight standard from Phenomenex was run as part of the analysis and the results were overlaid.

[0163] Hp(162-406) mutant HSA-fused Hp(162-406) variants were purified on the AKTAxpress system (Cytiva) using an automated method for tandem chromatography. Specifically, 1-2 L of Expi293F supernatant was loaded onto a 5 ml CaptureSelect HSA affinity column (Thermo) equilibrated in 10 mM Tris; 150 mM NaCl, pH 7.5. The bound HSA-fused protein was then washed with 10 mM Tris; 150 mM NaCl, pH 7.5 to reduce non-specific interacting proteins before elution into a retention loop using 20 mM Tris, pH 7.4. The captured eluate from the CaptureSelect HSA affinity column was then injected onto a Superdex 200 26 / 60 HiPrep size-exclusion column (Cytiva) for preparative separation of aggregates and size species in MT-PBS mobile phase.

[0164] Fractions containing proteins of the predicted size were pooled and concentrated using Amicon Ultra-15 centrifugal ultrafiltration devices (Merck-Millipore) prior to flow through a 0.22um filter. Protein concentration was then measured by OD280 using a Trinean DropSense96 system (Trinean) and purity was verified by SDS-PAGE separation on a NuPAGE 4-12% Bis-Tris gel (Thermo Fisher Scientific). Higher order species levels of the solutions were assessed using an analytical Superdex 200 Increase (15 / 50) size exclusion column interfaced to an Agilent 1260 Infinity HPLC with MT-PBS as the mobile phase. For comparison, 1ul of Aqueous SEC1 (AL0-3042) molecular weight standard from Phenomenex was run as part of the analysis and the results were overlaid.

[0165] B.7. Purification of Fc Fusion Proteins Hp(148-406) mutant Hp Fc fusion mutants were purified on the AKTAxpress system (Cytiva) using an automated method for tandem chromatography. Specifically, 30 ml of Expi293F supernatant was loaded onto a 1 ml MabSelect SuRe pcc column (Cytiva) equilibrated in MT-PBS. The bound Fc fusion protein was then washed with 500 mM L-Arg; 10 mM Tris; 150 mM NaCl (pH 7.5) to reduce aggregates and endotoxins before elution into a retention loop using 0.1 M sodium acetate (pH 3.0). The eluate captured from the MabSelect SuRe pcc column was then injected onto a Superdex 200 16 / 60 size exclusion column (Cytiva) equilibrated in MT-PBS to separate Hp molecular species by size. The captured eluate from the Mimetic Blue column was then injected onto a HiPrep 26 / 10 desalting column (Cytiva) for buffer exchange into MT-PBS.

[0166] Protein-containing fractions containing all size species were pooled and concentrated using Amicon Ultra-15 centrifugal ultrafiltration devices (Merck-Millipore, MS, USA) prior to flow through a 0.22um filter. Protein concentration was then measured by OD280 using a Trinean DropSense96 system (Trinean) and purity was verified by SDS-PAGE separation on NuPAGE 4-12% Bis-Tris gels (Thermo Fisher Scientific). Higher order species levels of the solutions were assessed using a Superdex 200 Increase (15 / 50) size exclusion column connected to an Agilent 1260 Infinity HPLC with MT-PBS as the mobile phase. For comparison, 1ul of Aqueous SEC1 (AL0-3042) molecular weight standard from Phenomenex was run as part of the analysis and the results were overlaid.

[0167] Hp(162-406) mutant Hp Fc fusion mutants were purified on the AKTAxpress system (Cytiva) using an automated method for tandem chromatography. Specifically, 30 ml of Expi293F supernatant was loaded onto a 5 ml MabSelect SuRe pcc column (Cytiva) equilibrated in MT-PBS. The bound Fc fusion protein was then washed with 500 mM L-Arg; 10 mM Tris; 150 mM NaCl (pH 7.5) to reduce aggregates and endotoxins before elution into a retention loop using 0.1 M sodium acetate (pH 3.0). The eluate captured from the MabSelect SuRe pcc column was then injected onto a Superdex 200 16 / 60 size exclusion column (Cytiva) equilibrated in MT-PBS to separate Hp molecular species by size. The captured eluate from the Mimetic Blue column was then injected onto a Superdex 200 26 / 60 HiPrep size exclusion column (Cytiva) for preparative separation of aggregates and size species in MT-PBS mobile phase.

[0168] Fractions containing proteins of the predicted size were pooled and concentrated using Amicon Ultra-15 centrifugal ultrafiltration devices (Merck-Millipore) prior to flow through a 0.22um filter. Protein concentration was then measured by OD280 using a Trinean DropSense96 system (Trinean) and purity was verified by SDS-PAGE separation on a NuPAGE 4-12% Bis-Tris gel (Thermo Fisher Scientific). Higher order species levels of the solutions were assessed using an analytical Superdex 200 Increase (15 / 50) size exclusion column interfaced to an Agilent 1260 Infinity HPLC with MT-PBS as the mobile phase. For comparison, 1ul of Aqueous SEC1 (AL0-3042) molecular weight standard from Phenomenex was run as part of the analysis and the results were overlaid.

[0169] B.8. Qualitative Measurement of Hemoglobin Binding to Novel Proteins Hb-binding proteins were incubated with human hemoglobin (HbA) at different concentrations for 1 h at 37°C. Hp-bound and non-bound fractions of Hb (cell-free Hb) were determined by SEC-high performance liquid chromatography (SEC-HPLC) using an Ultimate 3000SD HPLC coupled to an LPG-3400SD quaternary pump and a photodiode array detector (DAD) (ThermoFisher). Plasma samples and Hb standards were separated on a Diol-300 (3 μm, 300 × 8.0 mm) column (YMC CO Ltd.) with PBS, pH 7.4 (Bichsel) as the mobile phase at a flow rate of 1 mL / min. Two wavelengths (λ = 280 nm and λ = 414 nm) were recorded for all samples. Bound and unbound Hb in plasma were determined by calculating the peak areas of both peaks (Hb: 6 min retention time for Hp; 8 min retention time for cell-free Hb).

[0170] B.9. Biotinylation of Haptoglobin Biotinylation was performed with the EZ-Link™ NHS-PEG Solid Phase Biotinylation Kit (Model: 21450; Thermo Scientific) according to the manufacture's protocol. Briefly, proteins were diluted in PBS to a concentration between 0.5-0.2 mg / ml. Distilled water was added to NHS-PEG4-Biotin to make a 1 mM solution. The following was added to each protein of interest:

number

[0171] The reaction was mixed quickly and incubated at room temperature for 30 minutes. The reaction was stopped by removing the excess biotin reagent via centrifugation at 1000×g for 2 minutes three times using an equilibrated desalting column. A new collection tube was attached and the biotinylated sample was slowly applied to the center of the dense resin bed at 1000×g for 2 minutes to collect the sample. The protein concentration was calculated.

[0172] B.10. Quantitative Measurement of Hemoglobin Binding to Novel Proteins Streptavidin pre-coated biosensors (model no. 18-5019; ForteBio) were used. The different Hp variants were biotinylated as described above and immobilized in assay buffer (PBS, 0.01% BSA, 0.002% Tween 20) at the indicated concentrations for each experiment. Hp variants were diluted in assay buffer (PBS, 0.01% BSA, 0.002% Tween 20). Association / dissociation kinetics analysis for Hp variants was performed at the indicated Hb concentrations for each experiment. The settings for each binding step were as shown in Table 2 (Table 2: Hu Haptoglobin 2FS(148-406)-8His as an example for Hu Haptoglobin 2FS(148-406)-8His). * The experimental setup of OctetRED96 for kinetic evaluation used for the experiments was chosen as shown in "Experimental setup of OctetRED96 for kinetic evaluation used for the experiments". A control for reference (sensor loaded with ligand but without analyte) was included in every experiment. Data were acquired at 30°C on an OctetRED96 (ForteBio) with the following settings:

[0173] [Table 3]

[0174] Data were analyzed by Data Analysis Software (ForteBio, Version 9.0). Data were processed by performing baseline alignment against the y-axis, stepwise correction, reference sensor subtraction, and curve smoothing via Savitzky-Golay filtering. Processed kinetic data sets were globally fitted using a 1:1 binding model. The accuracy of the fit was evaluated using parameters that describe how well the measured results resemble those calculated from the model used to analyze the data, Chi2 and R 2 Described by:

[0175] B.11. Measuring heme binding to novel proteins The heme binding method was described in Lipiski, 2013, with some adaptation. Briefly, heme-albumin (12.5 μM in PBS) was incubated with a heme-binding protein (e.g., human hemopexin). To follow the transfer of heme-albumin to heme-Hpx over time, a series of UV-VIS spectra were recorded (350-650 nm) using a Cary 60 UV-VIS Spectrophotometer (Agilent Technologies). For each time point, the concentrations of heme-albumin and heme-Hpx in the reaction mixture were resolved by full-spectrum deconvolution by applying the Lawson-Hanson NNLS (Non Negative Least Squares) algorithm (www.scipy.org) in SciPy. The heme loss rates (fast and slow) from met-Hb were determined by nonlinear regression using R (r-project.org) with the following biexponential model:

number

[0176] B.12. Binding to the CD163 Clearance Receptor by BLI Streptavidin pre-coated biosensors (model number: 18-5019; ForteBio) were used. Biotinylated human CD163 receptor was immobilized in assay buffer (PBS, 0.01% BSA, 0.002% Tween 20) at the concentrations indicated in each experiment. Hp:hemoglobin complexes were immobilized in assay buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 25 mM CaCl 2 , 0.05% Tween 20, 0.1% BSA). Association / dissociation kinetic analysis for haptoglobin:hemoglobin was performed at the indicated Hb concentrations for each experiment. Settings for each binding step were chosen as indicated in the table. A control for reference (sensor loaded with ligand but no analyte) was included in every experiment. Data were acquired at 30° C. on an OctetRED96 (ForteBio) with the following settings in Table 3:

[0177] [Table 4]

[0178] Data were analyzed by Data Analysis Software (ForteBio, Version 9.0). Data were processed by performing baseline alignment against the y-axis, stepwise correction, reference sensor subtraction, and curve smoothing via Savitzky-Golay filtering. Processed kinetic data sets were globally fitted using a 1:1 binding model. The accuracy of the fit was evaluated using parameters that describe how well the measured results resemble those calculated from the model used to analyze the data, Chi2 and R 2 Described by:

[0179] B.13. Binding to LRP1 Clearance Receptor Fragments by BLI Streptavidin pre-coated biosensors (model number: 18-5019; ForteBio) were used. Biotinylated LRP1 / CD91 domain 3 was immobilized at a concentration of 15 μg / mL in assay buffer (PBS, 0.1% BSA, 0.02% Tween 20). Heme-Hpx complex was diluted in assay buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 25 mM CaCl2, 0.05%, 0.1% BSA, Tween 20). Association / dissociation kinetic analysis for heme-hemopexin complex was performed at the indicated concentrations for each experiment. Settings for each binding step were chosen as shown in Table 4. A control for reference (sensor loaded with ligand but without analyte) was included in every experiment. Data were acquired at 30° C. on an OctetRED96 (ForteBio) with the following settings:

[0180] [Table 5]

[0181] Data were analyzed by Data Analysis Software (ForteBio, Version 9.0). Data were processed by performing baseline alignment against the y-axis, stepwise correction, reference sensor subtraction, and curve smoothing via Savitzky-Golay filtering. Processed kinetic data sets were globally fitted using a 1:1 binding model. The accuracy of the fit was evaluated using parameters that describe how well the measured results resemble those calculated from the model used to analyze the data, Chi2 and R 2 Described by:

[0182] B.14. Acceptance Criteria for BLI Experiments For accurate kinetic fitting, a maximum of one data point (out of a total of seven data points) was excluded from the calculations to achieve the criteria in Table 5.

[0183] [Table 6]

[0184] C. Results EXAMPLES

[0185] Amino acid sequence and processing of wild-type human haptoglobin. Hp was synthesized as a single polypeptide chain (proHp) that was proteolytically processed in the endoplasmic reticulum via complement C1r-like protein into an α subunit (9 kDa) and a β subunit (33 kDa), which were linked via disulfide bonds to form Hp monomers. Each Hp monomer protein could bind one Hb α-β dimer (K d to 10 -15 (as Hp1 and Hp2). Deoxygenated Hb does not bind to Hp. In humans, Hp exists as two allelic forms, Hp1 and Hp2, which differ only within their respective alpha chains, i.e., the beta chain is invariant. The Hp2 allele arises from the Hp1 allele by duplication of exon 3 with exon 4 (YangF et al., 1983, PNAS, 80(219):5875-5879). The Hp1 allele is further subdivided into Hp1F and Hp1S, which differ by two amino acids in the alpha chain: Asp52Asn, Lys53Glu, with the numbering convention used herein being Hp1F=D69 K70 and Hp1S=N69 E70 (van der Straten A et al., 1984, FEBS Lett., 168:103-107). The structure of Hp is shown in FIG. 1. EXAMPLES

[0186] Production of Hu haptoglobin beta chain protein in mammalian cells Hu haptoglobin (162-406)-8His, and Hu haptoglobin 2FSβ (162-406, C266A)-8His To generate the beta fragment of Hp produced in mammalian cells, a cDNA construct, Huhaptoglobin(162-406)-8His, was designed in which the beta fragment of human Hp begins immediately after the C1rLP cleavage site at amino acid 162 in the Hp 2FS polypeptide chain (Figure 3A, Figure 4A). An additional mutant, Huhaptoglobin2FSβ(162-406, C266A)-8His (C266A, Figure 4A), in which the unpaired cysteine ​​at amino acid 266 was mutated to an alanine, was also generated. Transient transfection of these expression constructs into Expi293F cells failed to generate any protein, indicating that the structure of the β chain was disrupted and therefore unstable in mammalian cells (Figure 4B).

[0187] HuHaptoglobin 2FS(148~406)-8His We generated a human Hp beta fragment construct, Hu haptoglobin 2FS(148-406)-8His, encoding amino acids 148-406, which retains the C1rLP cleavage site and the cysteines required for intrachain disulfide bonds (Figure 4B), and transfected these into Expi293F cells along with a construct encoding C1rLP, which allows processing of the remaining N-terminal amino acids of the alpha chain. Processing was retained to allow for the creation of future proteins in which the fusion partner is placed N-terminal to the beta fragment and linked via an interdomain disulfide bond. Figure 4C shows that robust expression of Hu haptoglobin 2FS(148-406)-8His was observed in contrast to Hu haptoglobin(162-406)-8His and Hu haptoglobin 2FSβ(162-406, C266A)-8His. Size-exclusion chromatography analysis of the purified culture supernatant using a Superdex 200 Increase 5 / 150 column (nickel affinity chromatography combined with an additional desalting step) indicated that the protein was homogenous without aggregation (Figure 4D). Protein purity and proper processing were verified by SDS-PAGE analysis (Figure 4D). EXAMPLES

[0188] Production of Hu haptoglobin beta chain protein variants with N-terminal or C-terminal fusion partners in mammalian cells A series of proteins were generated encoding the hu haptoglobin beta chain (162-406 or 148-406) fused at the N- or C-terminus to human hemopexin (Hpx), Hpx+mouse serum albumin (MSA), or human Hpx+Fc, human serum albumin (HSA), mouse serum albumin (MSA), or the Fc domain of mouse IgG2a (Figures 5A and 5B). Processing was preserved to allow for future generation of proteins in which the fusion partner is located N-terminal to the beta fragment (amino acids 148-406) and linked via an interdomain disulfide bond.

[0189] Creation of Hemopexin-Hu Haptoglobin beta Fusion Protein A series of constructs were generated containing human hemopexin (Hpx; amino acids 1-462; SEQ ID NO:12) at the N-terminus followed by a Gly-Ser linker and then fused to a human Hp beta fragment corresponding to amino acids 162-406 of SEQ ID NO:1 (FIG. 6Ai); a human Hp beta fragment corresponding to amino acids 162-406 of SEQ ID NO:1 in which the unpaired cysteine ​​at the position corresponding to amino acid residue 266 of SEQ ID NO:1 has been mutated to alanine (FIG. 6Aii); and a human Hp beta fragment corresponding to amino acids 148-406 of SEQ ID NO:1 (FIG. 6Aiii), which retains the C1r-LP cleavage site (SEQ ID NO:4) and the cysteine ​​required for intrachain disulfide bond formation. Constructs containing amino acids 148-406 of haptoglobin were co-transfected into Expi293F cells at a ratio of 90:10 between the Hp alpha chain and a construct encoding C1r-LP, which allows processing at the junction with the Hp beta chain. Figure 6B shows that robust expression of construct Huhemopexin-Huhaptoglobin2FS(148-406)-His and proteolytic cleavage by C1r-LP at the predicted site were observed, in contrast to Hpx constructs containing Huhaptoglobin(162-406) or Huhaptoglobin(162-406,C266A). Analytical SEC (nickel affinity chromatography followed by desalting) of purified culture supernatants indicated that Huhemopexin-Huhaptoglobin2FS(148-406)-His was produced as a homogenous protein of the predicted size with dramatically reduced aggregation (Figure 6Cii), compared to the broad peak observed for Huhemopexin(1-462)-Huhaptoglobin(162-406)-8His (Figure 6Ci). Protein purity and proper processing were verified by reducing and non-reducing SDS-PAGE analysis (FIG. 6Ciii).

[0190] Creation of HSA-Hu haptoglobin beta fusion protein A series of constructs were generated that contained human serum albumin (HSA) in the N-terminal linker and then fused to a human Hp beta fragment encoding amino acids 162-406 with an intervening 13xGly-Ser linker (Figure 7Ai); a human Hp beta fragment encoding amino acids 162-406 in which the unpaired cysteine ​​at amino acid 266 was mutated to an alanine and an intervening 13xGly-Ser linker (Figure 7Aii); a human Hp beta fragment encoding amino acids 148-406, retaining the C1r-LP cleavage site and the cysteine ​​required for intrachain disulfide bonding (Figure 7Aiii), and these were transfected into Expi293F cells along with a construct encoding C1r-LP, which allows processing of the remaining N-terminal amino acids of the alpha chain for constructs containing this site. Figure 7B shows that, in contrast to HSA constructs containing Huhaptoglobin(162-406) or Huhaptoglobin(162-406,C266A), robust expression of construct HSA-Huhaptoglobin2FS(148-406) and proteolytic cleavage by C1r-LP at the predicted site were observed. Analytical SEC analysis of purified culture supernatants using a Superdex 200 Increase 5 / 150 column (nickel affinity chromatography combined with an additional desalting step) indicated that HSA-Huhaptoglobin2FS(148-406) was homogenous with negligible aggregation (Figure 7Cii). In contrast, the preparative SEC chromatograms show that HSA-GS13-Hu haptoglobin (162-406) of the expected size (indicated by the arrow) was very low in abundance, and most of the material produced was multimerized or aggregated (Figure 7Ci). Protein purity and proper processing were verified by reducing and non-reducing SDS-PAGE analysis (Figure 7Ciii).

[0191] Creation of Fc-Hu haptoglobin beta fusion protein Constructs containing human IgG1Fc at the N-terminus fused to a human Hp beta fragment encoding amino acids 162-406 (FIG. 8Ai); and mouse IgG2aFc fused to a human Hp beta fragment encoding amino acids 148-406, which retains the C1r-LP cleavage site and the cysteine ​​required for intrachain disulfide bonds (FIG. 8Aii); were transfected into Expi293F cells together with a construct encoding C1rLP, which allows processing of the remaining N-terminal amino acids of the α chain for constructs containing this site. FIG. 8B shows that in contrast to the Fc construct containing Hu haptoglobin (162-406), expression of the construct HSA-Hu haptoglobin 2FS (148-406) and proteolytic cleavage by C1r-LP at the predicted site were observed. No protein was expressed or purified from the HuIgG1Fc-HuHaptoglobin(162-406) construct. In contrast, muIgG2aFc-HuHaptoglobin2FS(148-406) was robustly expressed, and analytical SEC of the correctly processed and affinity purified material by C1r-LP revealed a major peak of the predicted size for the Fc dimer. However, the expressed fusion protein was not homogenous and contained both high and low molecular weight species (Figure 8Cii).

[0192] Creation of Hemopexin-MSA-Hu Haptoglobin beta Fusion Protein Constructs were created that contained human hemopexin (Hpx; amino acids 1-462) at the N-terminus followed by mouse serum albumin (MSA) and then fused to a human Hp beta fragment encoding amino acids 162-406 (Figure 9Ai); a human Hp beta fragment encoding amino acids 162-406, or a human Hp beta fragment encoding amino acids 148-406 (Figure 9Aii), which retains the C1r-LP cleavage site and the cysteine ​​required for intrachain disulfide bonding; these were transfected into Expi293F cells along with a construct encoding C1rLP, which for constructs containing this site allows processing of the remaining N-terminal amino acids of the alpha chain. FIG. 9B shows that, in contrast to the Hpx construct containing Huhaptoglobin(162-406), robust expression of the construct Huhemopexin-msa-Huhaptoglobin2FS(148-406) and proteolytic cleavage by C1r-LP at the predicted site was observed. Preparative SEC on Huhemopexin-msa-Huhaptoglobin(162-406) showed that it was in extremely low yield with a large proportion of higher order species (FIG. 9Ci). In contrast, analytical SEC on purified culture supernatant (Mimetic Blue affinity followed by desalting) indicated that Huhemopexin-msa-Huhaptoglobin2FS(148-406) was produced as a homogenous protein of the predicted size (FIG. 9Cii). The purity and processing of purified Hu hemopexin-msa-Hu haptoglobin 2FS(148-406) were verified by reducing and non-reducing SDS-PAGE analysis (FIG. 9Ciii).

[0193] Creation of Hemopexin-Fc-Hu Haptoglobin beta Fusion Protein We generated constructs containing human hemopexin (Hpx; amino acids 1-462) at the N-terminus followed by a Gly-Ser linker, mouse IgG2aFc, and then fused to a human Hp beta fragment encoding amino acids 148-406, which retains the C1r-LP cleavage site and the cysteine ​​required for intrachain disulfide bonds (Figure 10A), and transfected these into Expi293F cells along with a construct encoding C1rLP, which allows processing of the remaining N-terminal amino acids of the alpha chain for constructs containing this site. Figure 10B shows that the construct containing Hu haptoglobin (162-406) demonstrated high Hu haptoglobin (162-406) expression and proteolytic cleavage by C1r-LP at the predicted site. Size exclusion chromatography analysis of purified culture supernatants using a Superdex 200 Increase 5 / 150 column (MabSelect SuRe PCC affinity chromatography combined with an additional desalting step) indicated that the protein made from Huhemopexin-mIgG2aFc-Huhaptoglobin2FS(148-406) was not homogenous and contained a large proportion of aggregates (Figure 10Ci), which were also visible on Western blot (Figure 10B). The purity and processing of purified Huhemopexin-mIgG2aFc-Huhaptoglobin2FS(148-406) were verified by reducing and non-reducing SDS-PAGE analysis (Figure 10Cii). EXAMPLES

[0194] Measurement of hemoglobin binding Mutants encoding amino acids 148-406 of the Hp beta fragment were evaluated for binding to hemoglobin (due to poor expression and protein aggregation, no mutants containing the wild-type beta fragment (amino acid residues 162-406 of SEQ ID NO:1) were tested for binding to hemoglobin).

[0195] Qualitative binding to hemoglobin by SEC The following mutants were qualitatively analyzed for their ability to bind hemoglobin: Huhaptoglobin2FS(148-406)-8His; Huhemopexin-Huhaptoglobin2FS(148-406)-8His; HSA-Huhaptoglobin2FS(148-406)-8His; muIgG2aFc-Huhaptoglobin2FS(148-406)-8His; Huhemopexin-msa-Huhaptoglobin2FS(148-406)-8His; and Huhemopexin-mIgG2aFc-Huhaptoglobin2FS(148-406)-His.

[0196] In a first step, all of the above recombinant mutants were qualitatively analyzed in relation to binding to Hb. Briefly, the recombinant mutants were incubated with different concentrations of human hemoglobin for 1 h at 37° C. The samples were separated on a SEC column (Diol-300; 3 μm, 300×8.0 mm) and the absorbance was recorded at 405 nm. As shown in FIG. 11, the HPLC trace for hemoglobin (blue line) shifted to the left when incubated with Hb-binding mutants, indicating an increase in size (red line) compared to the HPLC trace for Hb alone. Based on this qualitative binding evaluation, all Hp beta fragments (148-406) are capable of binding to hemoglobin independently of the fusion protein.

[0197] Quantitative binding to hemoglobin by BLI In humans, plasma haptoglobin (Hp) binds hemoglobin with high affinity. For quantitative assessment, Hp-mediated Hb binding was determined for the following Hp variants and data were compared to human plasma-derived Hp1-1: Hu haptoglobin 2FS(148-406)-8His; Hu hemopexin-Hu haptoglobin 2FS(148-406)-8His; and Hu haptoglobin 1-1 (plasma-derived).

[0198] The biotinylated mutants were immobilized on a streptavidin-coated biosensor and evaluated for binding to Hb. As shown in Table 6, binding to human Hp1-1 (plasma derived) results in a high affinity interaction (144 pM ± 39). For the Hp beta fragment alone (Hu haptoglobin 2FS(148-406)-8His), the K D Identical binding behavior was observed, with a k of 188 pM ± 42. Interestingly, the immobilized bifunctional SuperScavenger (HuHemopexin-HuHaptoglobin2FS(148-406)-8His) showed an almost two-fold faster on-rate (k on :4.4×10 5 1 / M seconds), and a slightly slower off-rate (k off :1.9×10 -5 The results showed an increase in binding affinity, which can be attributed to the increased binding affinity (1 / sec).

[0199] [Table 7] EXAMPLES

[0200] Measurement of heme binding Mutants containing a hemopexin domain (heme-binding domain) were evaluated for their heme-binding capacity and compared to wild-type hemopexin (plasma-derived hemopexin). Briefly, and as described above, each mutant was incubated with heme-albumin, which acts as a low-affinity heme donor similar to hemopexin. Spectra were recorded continuously for 5 hours, and the data were deconvoluted against reference spectra consisting of heme-albumin and hemopexin:heme. Using R Studio, the data was fitted with a bi-exponential model (described in Methods) and plotted as shown in FIG. 13. Table 7 summarizes the heme-binding capacity of the three tested mutants compared to plasma-derived hemopexin. All three mutants are believed to bind heme transferred from heme-albumin heme, as indicated by the red curves showing the concentration of heme bound to hemopexin at the indicated time points. Binding to heme is described as biexponential due to very rapid binding during the first few minutes followed by much slower binding behavior near saturation. This is true for all variants and is nearly equivalent to plasma-derived hemopexin. Interestingly, in relation to activity, all variants bind nearly 100 percent, except for the fusion protein containing mIg2aFc, which is about 80% active. The rate constants are summarized in the table.

[0201] [Table 8] EXAMPLES

[0202] Measurement of binding to CD163 Human CD163 (Hb scavenger receptor) is a 130 kDa glycoprotein that is expressed almost exclusively in cells of the monocyte lineage, with highest expression detected in macrophages in mature tissues, including Kupffer cells and red pulp macrophages. Structurally, CD163 belongs to the SRCR (scavenger receptor cysteine-rich) family of proteins, characterized by the presence of an SRCR domain in the extracellular region. CD163 is a natural high-affinity scavenger receptor for the hemoglobin-haptoglobin complex and is expressed at high levels primarily on monocytes and macrophages, and is therefore also used as a marker for cells derived from the monocyte / macrophage lineage. Under normal physiological conditions, Hp counters the toxicity of Hb by capturing released Hb and directing it to CD163-expressing macrophages, which internalize the complex.

[0203] As a consequence of the very similar binding behaviour to Hb for all tested Hp variants, complexes with hemoglobin were made and probed for their ability to bind to immobilised CD163 receptor compared to the haptoglobin 1-1 (plasma derived):hemoglobin complex.

[0204] The following mutants complexed with human hemoglobin: Hu haptoglobin 2FS(148-406)-8His; Hu hemopexin-Hu haptoglobin 2FS(148-406)-8His; and Hu haptoglobin 1-1 (plasma derived) were analyzed for their ability to bind to the CD163 receptor.

[0205] As shown in FIG. 14, both recombinant Hp variants were found to bind to immobilized CD163, albeit with different affinities, compared to the plasma-derived wild-type haptoglobin 1-1:hemoglobin complex. The binding behavior of both recombinant variants showed increased dissociation compared to Hp1-1:Hb, making it difficult to determine a reasonable KD using a 1:1 kinetic fitting model. Therefore, steady-state analysis was performed to estimate the kinetic constants (KD). As summarized in Table 8 below, both Huhaptoglobin2FS(148-406) and Huhemopexin-Huhaptoglobin2FS(148-406) complexed with hemoglobin had approximately 7-fold and 50-fold lower affinities compared to the haptoglobin:hemoglobin complex generated with Hp1-1. This observation is potentially explained by the presence of only one binding site (the binding site within the beta chain of Hp) compared to Hp1-1 purified from plasma.

[0206] [Table 9] EXAMPLES

[0207] Preservation of vascular nitric oxide signaling in the presence of hemoglobin A. Materials and Methods Vascular function assay Vascular function assays were performed using fresh porcine basilar arteries (n=20) obtained from a local slaughterhouse as described in Hugelhofer et al., 2019, J Clin Invest., 129(12):5219-5235. Briefly, after isolating the basilar artery, it was cut into 2 mm long sections. The vascular ring sections were then mounted onto the pins of a Multi-Channel Myograph System 620 M (Danish Myo Technology) and immersed in Krebs-Henseleit buffer. As previously described in Hugelshofer et al., 2020, J Vasc Res et al., 57:106-112, after stretching the vessels to reach an optimal passive pretension (IC1 with factor k = 0.80), 10 μM prostaglandin F2α (PGF2α; Sigma, Buchs, Switzerland) was added as a pre-contracting agent. This was followed by the addition of MAHMA-NONOate (ENZO Life Sciences) to induce NO-dependent vasodilation. For all vessels, the experiment consisted of three phases: first phase: NO-dependent vasodilation in Krebs-Henseleit buffer (KHB) in the absence of Hb (NO-dip); second phase: vasodilation after the addition of 10 μM Hb (dip); and third phase: vasodilation after the addition of an equimolar amount of haptoglobin (10 μM). The dilatation response recorded for each vessel was normalized to the NO-induced maximum dilatation without Hb exposure (initial vasodilation: equivalent to 100%) and to the level of tonic contraction before addition of MAHMA-NONOate (equivalent to 0%).

[0208] Hb and reconstituted lipoprotein (rLP) Hb for use in ex vivo experiments was purified from expired human blood concentrates as previously described (Elmer et al., 2011, J Chromatogr B Analyt Technol Biomed Life Sci., 879(2):131-138). Hb concentrations were determined spectrophotometrically with spectral deconvolution and are given as molar concentrations relative to the total heme amount, which is the equivalent amount of a single-chain subunit of Hb (α or β chain; 1 M Hb tetramer is equivalent to 4 M heme). For scavenger proteins (Hp; recombinant Hp constructs and hemopexin), 1 mole was considered to be the binding capacity equivalent to 1 mole of heme. For all Hb used in these studies, the ferrous-containing Hb (HbFe) determined spectrophotometrically was used. 2+ O 2 The fraction of rLP was obtained from CSL Behring, Bern, Switzerland.

[0209] Lipoprotein peroxidation assay The effectiveness of haptoglobin variants in preventing Hb oxidation was quantified by measuring the formation of malondialdehyde (MDA), an end product of lipid peroxidation, following incubation of Hb-haptoglobin complexes with rLP. 30 μL of 96-well plates containing rLP (2 mg / mL) and Hb or Hb complexed with haptoglobin variants (10 μM) were incubated at 37° C. for 4 hours. The concentration of MDA was then measured using the TBARS assay (Deuel et al., 2015, Free Radical Biology and Medicine, 89:931-943). Briefly, 125 μL of 750 mM trichloroacetic acid in 1 M HCl was added to the samples, followed by vortexing (5 seconds) followed by the addition of 100 μL of 25 mM 2-thiobarbituric acid in 1 M NaOH. TBARS was quantified in the supernatant after an incubation time of 60 min at 80° C. To achieve a more sensitive but relative quantification, fluorescence emission was measured at 550 nm using 510 nm as excitation wavelength.

[0210] B. Results Preservation of vascular nitric oxide signaling in ex vivo vascular function experiments depends on Hp size and fusion partner To evaluate the NO sparing capacity of the different Hp variants, we used a well-established ex vivo vascular function assay in which the rescue of NO-dependent vasodilatory responses after the addition of Hb scavengers was measured. In all experiments, the addition of 10 μM Hb to KHB resulted in an inhibition of vasorelaxation below control vasorelaxation without Hb by 10% (median relative relaxation: 9.7%). Subsequent addition of Hb scavengers resulted in the restoration of vasorelaxation in the presence of NO donors for all Hp variants (FIG. 15). In all experiments, human plasma-derived Hp2-2 was used as a reference / gold standard for comparison. In plasma Hp1-1, recHp1-1, and recHpCD163low groups, rescue was similar compared to Hp2-2, with no evidence of difference (Table 9). For the small molecular weight miniHp, rescue was less pronounced than that by Hp2-2 (median Hp2-2: 87.93%, median miniHp: 67.99%, p-value < 0.0001). Bifunctional constructs (SuperScavenger, Hpx-Hp constructs) showed intermediate rescue (median: 48.13%). This was due to the fact that Hb(αβ) 2 -Hp 1-1 complex, but smaller than Hb(αβ) 1 -Hb(αβ), which is larger than the miniHp complex 1 -This is likely related to the size of the Hp-Hpx complex.

[0211] [Table 10]

[0212] Prevention of lipid peroxidation is independent of Hp mutants To assess the antioxidant potential of recombinant haptoglobin variants, we measured the occurrence of MDA in mixtures of Hb and rLP, which contains unsaturated phosphatidylcholine, the main physiological lipid substrate for Hb peroxidation in vivo (Deuel et al., 2015; Free Radic Biol Med., 89:931-43). When Hb was mixed with equimolar amounts of Hp, no MDA was detected after 4 hours of incubation at 37°C, regardless of the Hp variant assessed (Figure 16A). We then repeated the experiment with increasing concentrations of Hb ranging from substoichiometric to superstoichiometric concentrations with respect to Hp (Figure 16B). In this experiment, we found that recombinant Hp variants prevented lipid peroxidation at concentrations up to Hb concentrations equimolar to Hp β chains. Even at excess concentrations of Hb over Hp, the concentration-oxidation relationship followed the same shape as for Hb alone. The only exception was observed with Hp-Hpx SuperScavenger, which showed a marked reduction in the occurrence of MDA even at supra-stoichiometric Hb concentrations, consistent with a heme-directed antioxidant function of Hpx (Deuel et al., 2015) that provided synergistic protection with Hp.

[0213] Binding of heme:Hx complexes with plasma-derived Hx and heme:Hx complexes with Hx-Hp fusion protein to LRP1 Complexation of Hx with heme leads to association with its scavenging receptor, CD91 / LRP1 (Hvidberg et al., 2005, Blood, 106(7):2572-9). Thus, in a final set of binding experiments, we explored the ability of Hx-Hp fusion proteins to bind to CD91 / LRP1 in comparison to plasma-derived Hx complexed with heme. Since the full-length receptor is difficult to recombinantly express, we immobilized only a fragment of CD91 / LRP1 (cluster III). In previous studies, we identified that of the four clusters, cluster III contains the binding site for heme:Hx (unpublished data). As shown in Table 10, we found that both heme complexes bound to the immobilized receptor fragments in a roughly equivalent manner, with KDs in the high nanomolar range. As illustrated in Figure 17, in contrast to Hx, which does not bind to LRP1 in the absence of heme, uncomplexed Hx-Hp showed binding affinity to immobilized LRP1, albeit at a very low level. Whether this is due to the artificial scaffold of the fusion protein remains to be explored.

[0214] [Table 11]

[0215] Consideration The inventors have previously shown that Hp can be expressed in high yields as a functional protein in a transient eukaryotic expression system, resulting in a molecule of appropriate size, structure, and function (Schaer, Owczarek et al., 2018, BMC Biotechnol., 18:15).

[0216] The major functions of Hp (binding by Hb and binding to CD163) are mediated by the Hp β chain (see Melamed-Frank, 2001, Blood, 98(13):3693-8; and Alayash, Andersen et al., 2013, Trends in Biotechnology, 31(1):2-3). To further characterize the minimal domain of haptoglobin required for binding to Hb, constructs were made in which the β chain of human Hp begins immediately after the C1r-LP cleavage site within the Hp polypeptide chain. Additional mutants were also made in which the unpaired cysteine ​​at amino acid 266 was mutated to an alanine. However, transient transfection of these expression constructs failed to produce protein, indicating that the structure of the β chain was disrupted and therefore unstable in mammalian cells. Unexpectedly, an alternative design construct in which the human Hpβ chain further contains 14 consecutive N-terminal amino acids of the Hpα chain, thereby retaining the C1r-LP cleavage site and the cysteine ​​required for intrachain disulfide bonds, resulted in robust and stable expression of functional Hpβ chain protein. The modified construct also allows the creation of fusion proteins in which a fusion partner (e.g., an additional functional moiety) linked to the Hpβ chain fragment via an interdomain disulfide bond is placed, for example, at the N-terminus of the N-terminally truncated Hp alpha chain. This is advantageous because it allows the creation of dual-targeting therapeutic molecules, an illustrative example of which includes the haptoglobin-hemopexin (Hp-Hpx) conjugate, which is used as a scavenger for both cell-free hemoglobin and cell-free heme.

Claims

1. 1. An expression system for producing a recombinant haptoglobin beta chain or a hemoglobin-binding fragment thereof in a mammalian cell, comprising: (a) a first nucleic acid sequence encoding an N-terminally truncated prohaptoglobin (proHp), the N-terminally truncated proHp comprising (i) at least 14 consecutive C-terminal amino acid residues of a haptoglobin alpha chain, and (ii) a haptoglobin beta chain, or a hemoglobin-binding fragment thereof, the N-terminally truncated proHp comprising an internal enzymatic cleavage site between the at least 14 consecutive C-terminal amino acid residues of the haptoglobin alpha chain and the haptoglobin beta chain, or a hemoglobin-binding fragment thereof; and (b) a second nucleic acid sequence encoding an enzyme capable of cleaving the N-terminal truncated proHp at the enzyme cleavage site; Including; An expression system in which a first nucleic acid sequence and a second nucleic acid sequence are introduced into a mammalian cell, and then, when the N-terminally truncated proHp and an enzyme are expressed in the cell, the enzyme is capable of cleaving the N-terminally truncated proHp at an internal enzyme cleavage site, thereby releasing the haptoglobin beta chain or a hemoglobin-binding fragment thereof from the N-terminally truncated proHp.

2. The expression system of claim 1 , wherein the proHp is human proHp.

3. a) human proHp comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:1; and / or b) the N-terminally truncated proHp comprises an amino acid sequence having at least 80% sequence identity with amino acid residues 148 to 406 of SEQ ID NO:1; and / or c) N-terminally truncated proHp comprises an amino acid sequence having at least 90% sequence identity with amino acid residues 148 to 406 of SEQ ID NO:1; and / or d) N-terminal truncated proHp has at least one amino acid sequence identical to amino acid residues 148-406 of SEQ ID NO:

1. and / or e) The expression system according to claim 2, wherein the N-terminally truncated proHp consists of amino acid residues 148 to 406 of SEQ ID NO:

1.

4. The expression system according to any one of claims 1 to 3, wherein the internal enzyme cleavage site is selected from the group consisting of a furin cleavage site, a serine protease cleavage site, a cysteine ​​protease cleavage site, an aspartic acid protease cleavage site, a metalloprotease cleavage site, and a threonine protease cleavage site.

5. The expression system of claim 4, wherein the serine protease cleavage site is a C1r-like protein (C1rLP) cleavage site or a functional variant thereof.

6. 6. The expression system according to claim 1, wherein the N-terminally truncated proHp comprises a disulfide bond between the 14 consecutive C-terminal amino acid residues of the Hp α chain and the Hp β chain.

7. N-terminally truncated proHp is a) a disulfide bond between a cysteine ​​residue among at least 14 consecutive C-terminal amino acid residues of the haptoglobin alpha chain and a cysteine ​​residue at a position corresponding to amino acid position 266 of SEQ ID NO:1; and / or b) a disulfide bond between a cysteine ​​residue at a position corresponding to amino acid position 149 and a cysteine ​​residue at a position corresponding to amino acid position 266 of SEQ ID NO:

1. The expression system of claim 6 .

8. The expression system according to any one of claims 1 to 7, wherein the N-terminally truncated proHp encoded by the first nucleic acid sequence comprises a further functional moiety, preferably a therapeutic agent.

9. 9. The expression system of claim 8, wherein the further functional moiety is selected from the group consisting of albumin, the Fc domain of an immunoglobulin or an FcRn-binding fragment thereof, and hemopexin or a heme-binding fragment thereof.

10. 10. The expression system of claim 8 or 9, wherein the further functional moiety is linked to one or more of the at least 14 consecutive C-terminal amino acid residues of the haptoglobin alpha chain.

11. 11. The expression system of claim 10, wherein the further functional moiety is linked to the N-terminus of at least 14 consecutive C-terminal amino acid residues of the haptoglobin alpha chain, preferably via a linker, more preferably via a peptide linker.

12. 12. The expression system according to any one of claims 1 to 11, wherein expression of the N-terminally truncated proHp in the mammalian cell is driven by a first mammalian regulatory sequence, operably linked to a first nucleic acid sequence, and expression of the serine protease in the mammalian cell is driven by a second mammalian regulatory sequence, operably linked to a second nucleic acid sequence, preferably the first mammalian regulatory sequence being different from the second mammalian regulatory sequence.

13. 13. The expression system according to any one of claims 1 to 12, wherein expression of the polypeptide in the mammalian cell is driven by a first mammalian regulatory sequence operably linked to a first nucleic acid sequence and expression of the serine protease in the mammalian cell is driven by a second mammalian regulatory sequence operably linked to a second nucleic acid sequence, preferably the first mammalian regulatory sequence being different from the second mammalian regulatory sequence.

14. 1. An expression vector for producing a recombinant haptoglobin beta chain or a hemoglobin-binding fragment thereof in a mammalian cell, comprising: (a) a first nucleic acid sequence according to any one of claims 1 to 13; and (b) a second nucleic acid sequence according to any one of claims 1 to 13.

1. An expression vector comprising:

15. a) the first nucleic acid sequence and the second nucleic acid sequence are operably linked to a common mammalian regulatory sequence; and / or b) the first nucleic acid sequence is operably linked to a first mammalian regulatory sequence and the second nucleic acid sequence is operably linked to a second mammalian regulatory sequence, the first mammalian regulatory sequence being different from the second mammalian regulatory sequence; The expression vector of claim 14.

16. A mammalian cell transfected or transduced with an expression system according to any one of claims 1 to 13, or with an expression vector according to claim 14 or 15.

17. 17. The mammalian cell of claim 16, which is a Chinese hamster ovary (CHO) cell or a human fetal kidney cell.

18. 1. A method for making a recombinant haptoglobin beta chain or a hemoglobin-binding fragment thereof, comprising: (a) introducing an expression system according to any one of claims 1 to 13, or an expression vector according to claim 14 or 15 into a mammalian cell to produce a transfected mammalian cell; (b) culturing the transfected mammalian cells of step (a) under conditions and for a period of time sufficient to permit production of a recombinant haptoglobin beta chain or a hemoglobin-binding fragment thereof by the transfected mammalian cells; and (c) recovering the recombinant haptoglobin beta chain or hemoglobin-binding fragment thereof produced in step (b). The method includes:

19. 19. The method of claim 18, wherein the cell is a CHO cell or a human fetal kidney cell.

20. 1. A recombinant hemoglobin-binding molecule comprising: (i) a haptoglobin beta chain or a hemoglobin-binding fragment thereof; and (ii) an N-terminally truncated haptoglobin alpha chain, wherein the N-terminally truncated haptoglobin alpha chain comprises at least 14 contiguous C-terminal amino acid residues of the haptoglobin alpha chain, and wherein the at least 14 contiguous C-terminal amino acid residues of the haptoglobin alpha chain are non-contiguous with the haptoglobin beta chain or a hemoglobin-binding fragment thereof, and wherein the N-terminally truncated haptoglobin alpha chain is joined to the haptoglobin beta chain or a hemoglobin-binding fragment thereof.

21. 21. The recombinant hemoglobin-binding molecule of claim 20, wherein the N-terminally truncated haptoglobin alpha chain is joined to the haptoglobin beta chain or hemoglobin-binding fragment thereof by a disulfide bond between a first cysteine ​​residue in the haptoglobin beta chain or hemoglobin-binding fragment thereof and a second cysteine ​​residue within at least 14 consecutive C-terminal amino acid residues of the haptoglobin alpha chain.

22. 22. A recombinant hemoglobin binding molecule according to claim 20 or claim 21, further comprising a further functional moiety.

23. Further functional parts are a) is joined to an N-terminal truncated haptoglobin alpha chain; and / or b) a heme-binding moiety selected from the group consisting of an Fc domain of an immunoglobulin or an FcRn-binding fragment thereof, and albumin, preferably hemopexin or a heme-binding fragment thereof; 23. The recombinant hemoglobin binding molecule of claim 22.

24. The recombinant hemoglobin binding molecule of claim 22 or 23, wherein the haptoglobin beta chain comprises an amino acid sequence having at least 80% sequence identity with amino acid residues 162 to 406 of SEQ ID NO:

1.

25. 20. A recombinant haptoglobin beta chain or a hemoglobin-binding fragment thereof produced by the method of claim 18 or 19.

26. A therapeutically effective amount of a recombinant haptoglobin beta chain or a hemoglobin-binding fragment thereof described in claim 25, or a recombinant hemoglobin-binding molecule described in claim 20 or 21, for use in the treatment or prevention of a condition associated with red blood cell lysis and release of cell-free hemoglobin (Hb) in a subject.

27. The condition is a) hemorrhagic stroke, preferably spontaneous, traumatic, intraventricular or subarachnoid hemorrhage, or b) Hemoglobinopathies, preferably sickle cell disease or α- or β-thalassemia 27. The recombinant haptoglobin beta chain or hemoglobin-binding fragment thereof for use according to claim 26,

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