Recombinant Heme Oxygenase-1 (HO-1) for the Treatment of Sickle Cell Disease

Abstract

The present invention provides, among other things, methods and compositions for making and using recombinant heme oxygenase for treating sickle cell disease. In some embodiments, recombinant heme oxygenase proteins are truncation variants, or Fc fusion proteins with increased half-life and/or reduced aggregation.

Claims

1. A method of treating sickle cell disease comprising administering to a subject in need of treatment a recombinant heme oxygenase-1 (rHO-1) protein.

2. The method of claim 1, wherein the rHO-1 protein comprises an amino acid sequence with at least 85% identity to residues 1-261 of SEQ ID NO:1.

3. The method of claim 1 or 2, wherein the rHO-1 protein comprises an amino acid sequence with at least 90% identity to residues 1-261 of SEQ ID NO:1.

4. The method of any one of the preceding claims, wherein the rHO-1 protein comprises an amino acid sequence with at least 95% identity to residues 1-261 of SEQ ID NO:1.

5. The method of any one of the preceding claims, wherein the rHO-1 protein comprises an amino acid sequence identical to residues 1-261 of SEQ ID NO:1.

6. The method of any one of the preceding claims, wherein the rHO-1 protein comprises SEQ ID NO:1.

7. The method of any one of claims 1-4, wherein the rHO-1 protein comprises K18, T21, H25, Y134, G143, L147, K179, and F207.

8. The method of claim 1, wherein the rHO-1 protein is truncated at the N-terminus at residue M9 of SEQ ID NO: 1.

9. The method of claim 7 or 8, wherein the rHO-1 protein is truncated at residues corresponding to K226, A233, R237, T261, and/or A265.

10. The method of claim 8, wherein the rHO-1 protein is truncated at the residue corresponding to K226.

11. The method of claim 1, wherein the rHO-1 protein comprises an amino acid sequence with at least 85% identity to residues 1-226 of SEQ ID NO:1.

12. The method of claim 1, wherein the rHO-1 protein comprises an amino acid sequence with at least 90% identity to residues 1-226 of SEQ ID NO:1.

13. The method of claim 1, wherein the rHO-1 protein comprises an amino acid sequence with at least 95% identity to residues 1-226 of SEQ ID NO:1.

14. The method of claim 1, wherein the rHO-1 comprises an amino acid sequence identical to residues 1-226 of SEQ ID NO:1.

15. The method of any one of the preceding claims, wherein the rHO-1 protein comprises an amino acid substitution at a position corresponding to 33.

16. The method of claim 15, wherein the rHO-1 protein comprises an F33L substitution.

17. The method of any one of the preceding claims, wherein the rHO-1 protein comprises an Fc domain fused to an rHO-1 protein domain.

18. The method of claim 17, wherein the N-terminus of the Fc domain is fused to the C-terminus of the rHO-1 protein domain.

19. The method of claim 17, wherein the C-terminus of the Fc domain is fused to the N-terminus of the rHO-1 protein domain.

20. The method of any one of claims 17-19, wherein the rHO-1 protein comprises a linker between the rHO-1 protein domain and the Fc domain.

21. The method of claim 20, wherein the linker comprises a sequence of (GGGGS).sub.4 (SEQ ID NO: 21).

22. The method of any one of claims 17-21, wherein the rHO-1 protein is a multimer comprising at least one monomer comprising an Fc domain fused to an rHO-1 protein domain.

23. The method of claim 22, wherein the multimer is a dimer.

24. The method of claim 22 or 23, wherein the multimer comprises a monomer comprising an Fc domain not fused to an rHO-1 protein domain.

25. The method of claim 22, wherein the rHO-1 protein is a dimer with one monomer comprising an Fc domain fused to an rHO-1 protein domain and another monomer comprising an Fc domain not fused to an rHO-1 protein domain.

26. The method of any one of claims 17-25, wherein the Fc domain comprises one or more mutations to enhance half-life, reduce aggregation and/or reduce the effector function.

27. The method of claim 26, wherein the one or more mutations comprise an amino acid substitution at one or more positions corresponding to 234, 235, 251, 252, 254, 255, 256, 308, 309, 311, 312, 314, 347, 349, 350, 351, 354, 360, 366, 385, 386, 387, 389, 392, 394, 399, 405, 407, 409, 428, 433, 434, 435, and 436 of IgG1 Fc domain.

28. The method of claim 27, wherein the one or more mutations comprises L234A and L235A amino acid substitutions.

29. The method of claim 27, wherein the one or more mutations comprise amino acid substitutions selected from L234A, L235A, Q347R, Y349C, T350V, L351Y, L351V, S354C, E356K, E357K, K360E, T366L, T366W, T366S, K370D, L368A, K392L, K392D, T394W, D399V, D399K, F405T, F405A, Y407V, K409W and K409D of IgG1 Fc domain.

30. The method of claim 27, wherein the one or more mutations on one monomer chain comprises K360E, K409W and Y349C amino acid substitutions and on a second monomer chain, Q347R, D399V, F405T and S354C amino acid substitutions.

31. The method of claim 27, wherein the one or more mutations on one monomer chain comprises T350V, L351Y, F405A and Y407V amino acid substitutions and on a second monomer chain, T350V, T366L, K392L and T394W amino acid substitutions.

32. The method of claim 27, wherein the one or more mutations comprises K360E, K409W, Y349C, Q347R, D399V, F405T and S354C amino acid substitutions.

33. The rHO-1 protein of claim 27, wherein the one or more mutations comprises T350V, L351Y, F405A, Y407V, T366L, K392L and T394W amino acid substitutions.

34. The method of any one of the preceding claims, wherein the rHO-1 protein is administered intravenously or subcutaneously.

35. The method of any one of the preceding claims, wherein administering the rHO-1 protein results in a reduced free heme level in plasma compared to a control.

36. The method of any one of the preceding claims, wherein administering the rHO-1 protein results in a reduced free heme level in plasma to below about 10 mM.

37. The method of any one of the preceding claims, wherein administering the rHO-1 protein results in an increased HO-1 activity in plasma compared to a control.

38. The method of any one of the preceding claims, wherein administering the rHO-1 protein results in an HO-1 activity in plasma at or above 10% of a normal serum HO-1 activity in a healthy individual.

39. The method of any one of the preceding claims, wherein administering the rHO-1 protein results in reduced or delayed onset of one of more symptoms including anemia, vasoocclusive crises (VOC), acute chest syndrome (ACS), pulmonary hypertension, or organ damage.

40. A recombinant heme oxygenase-1 (rHO-1) protein comprising an rHO-1 protein domain fused to an Fc domain.

41. The rHO-1 protein of claim 40, wherein the C-terminus of the rHO-1 protein domain is fused to the N-terminus of the Fc domain.

42. The rHO-1 protein of claim 40, wherein the N-terminus of the rHO-1 protein domain is fused to the C-terminus of the Fc domain.

43. The rHO-1 protein of any one of claims 40-42, wherein the rHO-1 protein comprises a linker between the rHO-1 protein domain and the Fc domain.

44. The rHO-1 protein of claim 43, wherein the linker comprises a sequence of GGGGS (SEQ ID NO: 10).

45. The rHO-1 protein of any one of claims 40-44, wherein the rHO-1 protein is a multimer comprising at least one monomer comprising an Fc domain fused to an rHO-1 protein domain.

46. The rHO-1 protein of claim 45, wherein the multimer is a dimer.

47. The rHO-1 protein of claim 45, wherein the multimer comprises a monomer comprising an Fc domain not fused to an rHO-1 protein domain.

48. The rHO-1 protein of claim 46 or 47, wherein the rHO-1 protein is a dimer with one monomer comprising an Fc domain fused to an rHO-1 protein domain and another monomer comprising an Fc domain not fused to an rHO-1 protein domain.

49. The rHO-1 protein of any one of claims 40-48, wherein the rHO-1 protein domain comprises an amino acid sequence with at least 85% identity to residues 1-261 of SEQ ID NO:1.

50. The rHO-1 protein of any one of claims 40-48, wherein the rHO-1 protein domain comprises an amino acid sequence with at least 90% identity to residues 1-261 of SEQ ID NO:1.

51. The rHO-1 protein of any one of claims 40-48, wherein the rHO-1 protein domain comprises an amino acid sequence with at least 95% identity to residues 1-261 of SEQ ID NO:1.

52. The rHO-1 protein of any one of claims 40-48, wherein the rHO-1 protein domain comprises an amino acid sequence identical to residues 1-261 of SEQ ID NO:1.

53. The rHO-1 protein of any one of claims 40-48, wherein the rHO-1 protein domain comprises SEQ ID NO:1.

54. The rHO-1 protein of any one of claims 40-53, wherein the rHO-1 protein domain comprises K18, T21, H25, Y134, G143, L147, K179, and F207.

55. The rHO-1 protein of claim 40, wherein the rHO-1 protein is truncated at the N-terminal at the residue corresponding to M9.

56. The rHO-1 protein of claim 54 or 55, wherein the rHO-1 protein domain further comprises K226, A233, R237, T261, and A265.

57. The rHO-1 protein of claim 56, wherein the rHO-1 is truncated at the residue corresponding to K226.

58. The rHO-1 protein of claim 40, wherein the rHO-1 protein comprises an amino acid sequence with at least 85% identity to residues 1-226 of SEQ ID NO: 1.

59. The rHO-1 protein of claim 40, wherein the rHO-1 protein comprises an amino acid sequence with at least 90% identity to residues 1-226 of SEQ ID NO:1.

60. The rHO-1 protein of claim 40, wherein the rHO-1 protein comprises an amino acid sequence with at least 95% identity to residues 1-226 of SEQ ID NO:1.

61. The rHO-1 protein of claim 40, wherein the rHO-1 comprises an amino acid sequence identical to residues 1-226 of SEQ ID NO:1.

62. The rHO-1 protein of any one of claims 40-48, wherein the rHO-1 protein domain comprises an amino acid substitution at a position corresponding to 33.

63. The rHO-1 protein of claim 62, wherein the rHO-1 protein domain comprises an F33L substitution.

64. The rHO-1 protein of any one of claims 40-63, wherein the Fc domain comprises one or more mutations to enhance half-life, reduce aggregation and/or reduce the effector function.

65. The rHO-1 protein of claim 64, wherein the one or more mutations comprise an amino acid substitution at one or more positions corresponding to 234, 235, 251, 252, 254, 255, 256, 308, 309, 311, 312, 314, 347, 349, 350, 351, 354, 360, 366, 385, 386, 387, 389, 392, 394, 399, 405, 407, 409, 428, 433, 434, 435, and 436 of IgG1 Fc domain.

66. The rHO-1 protein of claim 64, wherein the one or more mutations comprises L234A and L235A amino acid substitutions.

67. The rHO-1 protein of claim 64, wherein the one or more mutations on one chain comprises K360E, K409W and Y349C amino acid substitutions and on a second chain, Q347R, D399V, F405T and S354C amino acid substitutions.

68. The rHO-1 protein of claim 64, wherein the one or more mutations on one chain comprises T350V, L351Y, F405A and Y407V amino acid substitutions and on a second chain, T350V, T366L, K392L and T394W amino acid substitutions.

69. The rHO-1 protein of claim 64, wherein the one or more mutations comprises K360E, K409W, Y349C, Q347R, D399V, F405T and S354C amino acid substitutions.

70. The rHO-1 protein of claim 64, wherein the one or more mutations on one chain comprises T350V, L351Y, F405A, Y407V, T366L, K392L and T394W amino acid substitutions.

71. A nucleic acid encoding a recombinant heme oxygenase-1 (rHO-1) protein of any one of claims 40-70.

72. A cell comprising a nucleic acid encoding a recombinant heme oxygenase-1 (rHO-1) protein of any one of claims 40-70.

Description

BRIEF DESCRIPTION OF THE DRAWING

[0110] Drawings are for illustration purposes only; not for limitation.

[0111] FIG. 1A shows exemplary results of HO-1 enzymatic activity measured by nmole bilirubin product formed per mg of recombinant heme oxygenase per hour.

[0112] FIG. 1B shows exemplary results of heme oxygenase enzyme kinetics of 1-261-His and His-1-261 relative to 1-261-His (R&D) on a hemin substrate.

[0113] FIG. 2 shows exemplary results of pharmacokinetic analysis of 1-261-His in transgenic Townes sickle mice (SS mice).

[0114] FIG. 3 shows exemplary results of survival of transgenic SCD mice infused with purified hemin to induce ACS treated with 1-261-His or a vehicle control.

[0115] FIG. 4A shows exemplary results of real-time oxygen saturation of transgenic SCD mice infused with purified hemin to induce ACS treated with 1-261-His or a vehicle control.

[0116] FIG. 4B shows exemplary results of breath rate measurements of transgenic SCD mice infused with purified hemin to induce ACS treated with 1-261-His or a vehicle control.

[0117] FIG. 4C shows exemplary results of heart-rate measurements of transgenic SCD mice infused with purified hemin to induce ACS treated with 1-261-His or a vehicle control.

[0118] FIG. 4D shows exemplary results of pulse distention measurements of transgenic SCD mice infused with purified hemin to induce ACS treated with 1-261-His or a vehicle control.

[0119] FIG. 4E shows exemplary wet/dry weight ratios of the lungs of transgenic SCD mice infused with purified hemin to induce ACS treated with 1-261-His or vehicle control. S and D denotes mice that survived (S) or died (D) from the ACS.

[0120] FIG. 5A-5Q show schematics of HO-1 1-261-His and Fc fusion constructs V1-V16 generated for extended half-life and/or reduced aggregation. FIG. 5C discloses SEQ ID NO: 21, FIG. 5D discloses SEQ ID NO: 21. FIG. 5O discloses SEQ ID NO: 64, FIG. 5P discloses SEQ ID NO: 63 and FIG. 5Q discloses SEQ ID NO: 64.

[0121] FIG. 6 shows exemplary results of enzymatic activity of 1-261-Fc fusion forms relative to 1-261-His and also compares the activity of V2 and V3 aggregates.

[0122] FIG. 7 shows exemplary results of a pharmacokinetic profile of 1-261-Fc V2, V3 and V4 fusion constructs in SS mice relative to 1-261-His.

[0123] FIG. 8A shows exemplary results of survival of transgenic SCD mice infused with purified hemin to induce ACS and treated with 1-261-Fc V3, 1-261-Fc V4 or vehicle control.

[0124] FIG. 8B shows exemplary results of blood oxygen saturation levels (i.e. oxygen carrying hemoglobin) of transgenic SCD mice infused with purified hemin to induce ACS treated with 1-261-Fc V3, 1-261-Fc V4 or vehicle control.

[0125] FIG. 8C shows exemplary measurements of wet/dry weight ratios of the lungs of transgenic SCD mice infused with purified hemin to induce ACS and treated with 1-261-Fc V3, 1-261-Fe V4 or vehicle control

[0126] FIG. 9A shows exemplary results of heart rate measurements of transgenic SCD mice infused with purified hemin to induce ACS and treated with 1-261-Fc V3, 1-261-Fc V4 or vehicle control.

[0127] FIG. 9B shows exemplary breath rate measurements of transgenic SCD mice infused with purified hemin to induce ACS and treated with 1-261-Fc V3, 1-261-Fc V4 or vehicle control.

[0128] FIG. 9C shows exemplary pulse distention of transgenic SCD mice infused with purified hemin to induce ACS and treated with 1-261-Fc V3, 1-261-Fc V4 or vehicle control.

[0129] FIG. 10A shows exemplary results of survival of transgenic SCD mice infused with purified hemin to induce ACS and treated with different doses of rHO-1-Fc V4 or vehicle control.

[0130] FIG. 10B shows exemplary results of survival of transgenic SCD mice infused with purified hemin to induce ACS and treated with different doses of rHO-1-Fc V4 or vehicle control.

[0131] FIG. 10C shows exemplary results of blood oxygen saturation levels (i.e. oxygen carrying hemoglobin) of transgenic SCD mice infused with purified hemin to induce ACS treated with different doses of rHO-1-Fc V4 or vehicle control.

[0132] FIG. 10D shows exemplary measurements of wet/dry weight ratios of the lungs of transgenic SCD mice infused with purified hemin to induce ACS and treated with different doses of rHO-1-Fc V4 or vehicle control.

[0133] FIG. 11 shows a schematic of the UnaG assay to measure HO-1 enzyme activity with high sensitivity. HO-1 catalyzes the production of biliverdin from heme, which is converted by biliverdin reductase to bilirubin. UnaG is a fluorescent protein that binds bilirubin resulting in a complex that is measured by fluorescence emitted at ˜520 nm.

[0134] FIG. 12A shows a linear dose-response curve for HO-1 enzyme activity up to about 1000 nM HO-1 amounts as measured by the UnaG assay.

[0135] FIG. 12B shows that the UnaG assay is specific as fluorescence is obtained only in the presence of biliverdin reductase and rHO-1 enzyme. The assay is substantially free of background fluorescence. In the absence of rHO-1, there is no fluorescence observed due to lack of biliverdin substrate. In the absence of biliverdin reductase, there is no fluorescence observed as no bilirubin is available to bind UnaG.

[0136] FIG. 13A shows rHO-1 enzyme activity traces with catalase and P450 oxidoreductase (POR). FIG. 13B shows rHO-1 enzyme activity traces without catalase and POR.

DETAILED DESCRIPTION

[0137] The present invention provides, among other things, methods and compositions for treating sickle cell disease, using recombinant heme oxygenase-1 (HO-1) as a protein therapeutic. In some embodiments, administration of recombinant heme oxygenase may ameliorate symptoms of sickle cell disease in acute cases. In some embodiments, administration of recombinant heme oxygenase may provide a treatment modality for long-term prophylaxis.

[0138] Various aspects of the invention are described in detail in the following sections. The use of sections is not meant to limit the invention. Each section can apply to any aspect of the invention. In this application, the use of “or” means “and/or” unless stated otherwise.

Sickle Cell Disease

[0139] Sickle cell disease (SCD) is a hemolytic disease caused by a single base pair substitution in the β-globin gene on chromosome 11, resulting in an amino acid switch from glutamic acid to valine, in position six of the β-globin chain of hemoglobin. This results in polymerization of deoxygenated hemoglobin through hydrophobic interactions with other hemoglobin molecules forming rigid polymer aggregates within red blood cells (hemoglobin S), distorting their shape, shortening their lifespan from 120 days to 15 days and promoting intravascular hemolysis. When erythrocytes are lysed, extracellular hemoglobin (Hb) is released and oxidized from Fe.sup.2+ to ferric Fe.sup.3+ hemoglobin (methemoglobin) which releases heme into the circulation. Oxidation of Hb releases extracellular heme, promotes vascular inflammation and damage in SCD, including painful vaso-occlusive crises (VOC) and Acute Chest Syndrome (ACS). Heme levels in patients with severe symptoms could be up to 2.2 mM. (Ghosh et al., 2013 J Clin Invest. 2013:123(11):4809-4820).

[0140] Intravascular hemolysis in SCD patients cause oxidative damage and trigger an inflammatory cascade. Free heme increases expression of endothelial adhesion molecules and apoptotic markers, promoting attachment of activated leukocytes and RBCs to the vessel wall. Chronic hemolysis may lead to depressed red blood cell counts and anemia. Chronic anemia may lead to myocardial infarction and increased cardiac output. Infarction may lead to bone necrosis and joint destruction.

[0141] Vasoocclusion may lead to leg ulcers and myofascial syndrome, and results in stroke in about 25% of patients by age 45. SCD patients suffer complications such as vaso-occlusive crises (VOC) characterized by severe pain, caused by vascular occlusion resulting from attachment of rigid sickle RBCs, activated leukocytes, and possibly platelets to the underlying activated, damaged vascular endothelium.

[0142] Fat embolism or bone ischemia may lead to Acute Chest Syndrome (ACS). Acute chest syndrome (ACS) is a major pulmonary complication of sickle cell disease (SCD). It is typically preceded by acute vaso-occlusive crisis and acute hemolysis. ACS diagnosis is associated with decreasing hemoglobin (Hb) concentration, hypoxemia, and multilobular lung infiltration. Lung injury in ACS is characterized predominantly by edema formation. Danger-associated molecular pattern molecules derived from the lysis of erythrocytes may ultimately contribute to lung injury in ACS. Hemin (the oxidized prosthetic moiety of Hb) is a potent inflammatory agonist and activator of TLR4. Hemolysis inevitably results in the release of hemin into the extracellular space. This process is likely to be accelerated in SCD because of the enhanced auto-oxidation of sickle-cell oxyhemoglobin and the presence of free hemin at high concentrations (˜1 μM) inside sickle erythrocytes. Hemin has been shown to induce ACS in a mouse model.

[0143] Liver ischemia can lead to hepatic dysfunction. Renal failure can occur in about 20% of SCD patients. Impaired splenic function results in a high incidence of infection. Organ damage is a key cause of death is SCD patients. Opioids (e.g. morphine, hydromorphone, fentanyl) are commonly utilized to manage VOC pain in the U.S., which carry addictive risks and significant side effects.

[0144] In newborns, red blood cells contain large proportion of fetal hemoglobin, hemoglobin F which binds to oxygen with greater affinity than adult hemoglobin. After birth, adult hemoglobin, Hemoglobin A expression increases. In children with sickle cell anemia, defective hemoglobin S is produced and continues into adulthood. Due to an elevated fetal hemoglobin level at birth, patients typically do not exhibit signs of sickle cell disease until 6 months after birth. Other than vaso-occlusion, common symptoms of SCD are manifested as fatigue, exercise intolerance, and shortness of breath due to anemia. Pediatric patients may experience delayed growth and fever, while adults may present with ulcers or bone injury. Other symptoms include jaundice, frequent urination, and muscle weakness.

[0145] HO-1 gene promoter polymorphism linked with increased HO-1 activity is associated with reduced incidence of ACS among children with SCD (Bean et al., 2012). Children with multiple ACS episodes have significantly higher plasma heme levels in comparison to age-matched counterparts with no history of ACS (Adisa et al., 2013). This can lead to acute hemolysis, severe hypoxemia, and massive infiltration in lung. Biomarkers such as impact on arterial oxygen saturation (% SpO2), hemoglobin (Hb), total plasma heme (TPH), and bilirubin levels, lung-weight, histopathology and vaso-occlusion may be used in diagnosis. Only two SCD disease-modifying oral therapies, hydroxyurea (HU) and Endari (L-glutamine) are approved, and alternative long-term blood transfusions are the only option. Hydroxyurea was originally approved by the FDA only in adults in 1998, and for children over 2 years in December 2017. This helps red blood cells retain their shape and flexibility, reducing complications and the need for frequent transfusions. Common side effects of hydroxyurea include low blood counts, gastrointestinal symptoms, and loss of appetite. In addition, effectiveness of prophylactic hydroxyurea is limited by poor patient adherence due to variable effectiveness, side effects, and toxicity requiring frequent blood count monitoring. Endari (L-glutamine) was approved in July 2017 for patients 5 years and older to reduce acute complications of SCD, including sudden, severe pain called sickle cell crises. Common side-effects of Endari include constipation, nausea, headache, abdominal pain, cough, pain in the extremities, back pain and chest pain. Prophylaxis using L-glutamine has only modest clinical effect. Voxelotor has been approved for treatment of anemia, but it does not reduce incidence of VOCs. Crizanlizumab requires monthly IV infusion that may result in poor adherence. Bone marrow or stem cell transplants may be an option for younger patients with severe SCD, but this necessitates finding a matching bone marrow or stem cell donor and the associated risks of transplant surgery can be serious and potentially life-threatening. Regular blood transfusions are used frequently to treat anemia and prevent long-term complications. There is a need in the art for developing therapeutic molecules to treat patients having sickle cell disease and experiencing VOC.

Recombinant Heme Oxygenase-1

[0146] Heme oxygenase protects the endothelium and tissues against hemolysis and oxidative stress. Inducing HO-1 expression may protect tissues and cells against ischemia, oxidative stress, inflammation, transplant rejection, apoptosis and cell proliferation. Heme oxygenase 1 (HO-1) plays an important role in heme detoxification by degrading heme into iron, carbon monoxide, and biliverdin. HO-1 specifically targets and degrades free heme to cytoprotective/anti-inflammatory by-products. Because rHO-1 is based on an endogenous protein it may significantly minimize off-target effects.

[0147] In some embodiments, as used herein, recombinant heme oxygenase proteins suitable for the present invention include any wild-type and modified heme oxygenase proteins (e.g., heme oxygenase proteins with amino acid mutations, deletions, insertions, and/or fusion proteins) that retain substantial heme oxygenase biological activity. Typically, a recombinant heme oxygenase protein is produced using recombinant technology. However, heme oxygenase proteins (wild-type or modified) purified from natural resources or synthesized chemically can be used according to the present invention.

[0148] In some embodiments, a suitable recombinant heme oxygenase protein or a recombinant heme oxygenase fusion protein has an in vivo half-life of or greater than about 1 minute, 2 minutes, 10 minutes, 15 minutes, 30 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, or 24 hours. In some embodiments, a suitable recombinant heme oxygenase protein or a recombinant heme oxygenase fusion protein has an in vivo half-life of or greater than about greater than about 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 54 hours, or 60 hours. In some embodiments, a recombinant heme oxygenase protein has an in vivo half-life of between 0.5 and 24 hours, between 1 day and 10 days, between 1 day and 9 days, between 1 day and 8 days, between 1 day and 7 days, between 1 day and 6 days, or between 1 day and 5 days.

[0149] In some embodiments, presented herein are engineered recombinant heme oxygenase variants. In some embodiments, the engineered recombinant HO variants are fused to IgG Fc. In some embodiments, the engineered recombinant HO variants are fused to human IgG1 Fc.

[0150] In some embodiments, recombinant heme oxygenase protein described herein comprises SEQ ID NO: 1.

TABLE-US-00001 (SEQ ID NO: 1) MERPQPDSMPQDLSEALK.sub.18EAT.sub.21KEVH.sub.25TQAENAEFMRN FQKGQVTRDGFKLVMASLYHIYVALEEEIERNKESPVFAPVY FPEELHRKAALEQDLAFWYGPRWQEVIPYTPAMQRYVKRLHE VGRTEPELLVAHAY.sub.134TRYLGDLSG.sub.143GQVL.sub.147KKIAQK ALDLPSSGEGLAFFTFPNIASATKFK.sub.179QLYRSRMNSLEMT PAVRQRVIEEAKTAF.sub.207LLNIQLFEELQELLTHDT DQ PSR PGL QRASNKVQDSAPVETPRGKPPLN RSQ PLLRWVLTLSFLVATVAVGLYAM.

[0151] In some embodiments, the recombinant HO-1 comprises a heme binding pocket comprising residues 18, 21, 25, 134, 143, 147, 179, 207 of SEQ ID NO: 1. In some embodiments, the recombinant HO-1 is phosphorylated at residue S.sub.229 of SEQ ID NO: 1. In some embodiments, the recombinant HO-1 comprises a substitution at residue 261 to facilitate E coli expression. In some embodiments, residue 261 is a Threonine (T). In some embodiments, the recombinant HO-1 is truncated HO-1 expression at residues K.sub.226, A.sub.233, R.sub.237, A.sub.265. In some embodiments, recombinant HO-1 comprises an endoplasmic reticulum (ER) membrane binding region. In some embodiments, the ER membrane binding region comprises the amino acid sequence PLLRWVLTLSFLVATVAVGLYAM (SEQ ID NO: 40).

[0152] In some embodiments, recombinant heme oxygenase protein described herein comprises SEQ ID NO: 2.

TABLE-US-00002 (SEQ ID NO: 2) MERPQPDSMPQDLSEALK.sub.18EAT.sub.21KEVH.sub.25TQA ENAEFMRNFQKGQVTRDGFKLVMASLYHIYVALE EEIERNKESPVFAPVYFPEELHRKAALEQDLAFW YGPRWQEVIPYTPAMQRYVKRLHEVGRTEPELLV AHAY.sub.134TRYLGDLSG.sub.143GQVL.sub.147KKIAQKALDL PSSGEGLAFFTFPNIASATKFK.sub.179QLYRSRMNS LEMTPAVRQRVIEEAKTAF.sub.207LLNIQLFEELQE LLTHDTK.sub.226DQS.sub.229PSRA.sub.233PGLR.sub.237QRAS NKVQDSAPVETPRGKPPLNT.sub.261 HHHHHH.

[0153] In some embodiments, recombinant heme oxygenase protein described herein comprises SEQ ID NO: 3.

TABLE-US-00003 (SEQ ID NO: 3) MERPQPDSMPQDLSEALK.sub.18EAT.sub.21KEVH.sub.25TQA ENAEFMRNFQKGQVTRDGFKLVMASLYHIYVALE EEIERNKESPVFAPVYFPEELHRKAALEQDLAFW YGPRWQEVIPYTPAMQRYVKRLHEVGRTEPELLV AHAY.sub.134TRYLGDLSG.sub.143GQVL.sub.147KKIAQKALDL PSSGEGLAFFTFPNIASATKFK.sub.179QLYRSRMNS LEMTPAVRQRVIEEAKTAF.sub.207LLNIQLFEELQE LLTHDTK.sub.226DQS.sub.229PSRA.sub.233PGLR.sub.237QRAS NKVQDSAPVETPRGKPPLNT.sub.261.

[0154] In some embodiments, recombinant heme oxygenase comprises a sequence at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

[0155] In some embodiments, recombinant heme oxygenase comprises truncated HO-1, wherein the HO-1 comprises residues selected from the group consisting of 1-226, 1-229, 1-233, 1-239 and 1-265 of SEQ ID NO: 1. A truncated HO-1 lacking the ER membrane binding region is especially useful for the therapeutic application disclosed here. For example, a truncated HO-1 comprising residues 1-226 of SEQ ID NO: 1 or 1-261 of SEQ ID NO: 1 is suitable for practicing the claimed invention.

[0156] In some embodiments, recombinant heme oxygenase comprises truncated HO-1 comprising N-terminal deletions of about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues of SEQ ID NO: 1.

[0157] In some embodiments, recombinant heme oxygenase comprises truncated HO-1 comprising C-terminal deletions of about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues of SEQ ID NO: 1.

[0158] In some embodiments, recombinant heme oxygenase comprises truncated HO-1 comprising residues 10-225 of SEQ ID NO: 1. In some embodiments, recombinant heme oxygenase comprises truncated HO-1 comprising residues 10-226 of SEQ ID NO: 1. In some embodiments, recombinant heme oxygenase comprises truncated HO-1 comprising residues 10-261 of SEQ ID NO: 1. In some embodiments, recombinant heme oxygenase comprises truncated HO-1 selected from a group consisting of residues 10-226, 10-229, 10-233, 10-239 and 10-265.

[0159] In some embodiments, recombinant heme oxygenase protein comprises amino acid residues comprising K18, T21, H25, Y134, G143, L147, K179 and/or F207 (according to SEQ ID NO:1). In some embodiments, recombinant heme oxygenase protein comprises amino acid residues comprising K18, T21, H25, Y134, G143, L147, K179, F207 and further comprises K226, A233, R237, T261, and/or A265 (according to SEQ ID NO: 1). In some embodiments, recombinant heme oxygenase protein comprises one or more an amino acid substitutions wherein the amino acid substitution is F33L (according to SEQ ID NO:1).

[0160] HO-1 Fusion Proteins

[0161] It is contemplated that a suitable recombinant HO-1 protein can be in a fusion protein configuration. For example, a recombinant HO-1 protein suitable for the present invention may be a fusion protein between a HO-1 domain and another domain or moiety that typically can facilitate a therapeutic effect of HO-1 by, for example, enhancing or increasing stability, potency and/or delivery of HO-1 protein, or reducing or eliminating immunogenicity, or clearance. Such suitable domains or moieties for a HO-1 fusion protein include but are not limited to Fc domain, XTEN domain, or human albumin fusions.

[0162] Fc Domain

[0163] In some embodiments, a suitable recombinant HO-1 protein comprises an Fc domain or a portion thereof that binds to the FcRn receptor. As a non-limiting example, a suitable Fc domain may be derived from an immunoglobulin subclass such as IgG. In some embodiments, a suitable Fc domain is derived from IgG1, IgG2, IgG3, or IgG4. In some embodiments, a suitable Fc domain is derived from IgM, IgA, IgD, or IgE. Particularly suitable Fc domains include those derived from human or humanized antibodies. In some embodiments, a suitable Fc domain is a modified Fc portion, such as a modified human Fc portion.

[0164] In some embodiments, a suitable Fc domain comprises an amino acid sequence as provided in Table 1.

TABLE-US-00004 TABLE 1 Exemplary Fc domains Sequence ID No. (description) Fc Domain* SEQ ID NO: 15 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD (wild- type GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK human IgG1 Fc) GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGR SEQIDNOT6 DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD (human IgG1 Fc- GVEVHNARTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK LALA) GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 17 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD (human IgG1 Fc- GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIERTISKAK NHance) GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALKFHYTQKSLSLSPGK SEQ ID NO: 18 DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD (human IgG1 Fc- GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIERTISKAK LALA + NHance) GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALKFHYTQKSLSLSPGK SEQ ID NO: 19 EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 20 KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK *LALA and NHance mutations are underlined.

[0165] In some embodiments, a suitable Fc domain comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous or identical to SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20.

[0166] It is contemplated that improved binding between the Fc domain and the FcRn receptor results in prolonged serum half-life of the recombinant protein. Thus, in some embodiments, a suitable Fc domain comprises one or more amino acid mutations that lead to improved binding to FcRn. Various mutations within the Fc domain that effect improved binding to FeRn are known in the art and can be adapted to practice the present invention. In some embodiments, a suitable Fc domain comprises one or more mutations at one or more positions corresponding to Thr 250, Met 252, Ser 254, Thr 256, Thr 307, Glu 380, Met 428, His 433 and/or Asn 434 of human IgG1, according to EU numbering.

[0167] In some embodiments, a suitable Fc domain comprises one or more mutations at one or more positions corresponding to L234, L235, H433 and N434 of human IgG1, according to EU numbering.

[0168] The Fc portion of a recombinant fusion protein may lead to targeting of cells that express Fc receptors leading to pro-inflammatory effects. Some mutations in the Fc domain reduce binding of the recombinant protein to the Fc gamma receptor and thereby inhibit effector functions. In one embodiment, effector function is antibody-dependent cell-mediated cytotoxicity (ADCC). For example, a suitable Fc domain may contain mutations of L234A (Leu234Ala) and/or L235A (Leu235Ala) (EU numbering). In some embodiments the L234A and L235A mutations are also referred to as the LALA mutations. As a non-limiting example, a suitable Fc domain may contain mutations L234A and L235A (EU numbering). An exemplary Fc domain sequence comprising the L234A and L235A mutations is shown as SEQ ID NO:16 in Table 1.

[0169] In some embodiments, a suitable Fc domain may contain mutations of H433K (His433Lys) and/or N434F (Asn434Phe) (EU numbering). As a non-limiting example, a suitable Fe domain may contain mutations H433K and N434F (EU numbering). In some embodiments the H433K and N434F mutations are also referred to as the NHance mutations. An exemplary Fc domain sequence incorporating the mutations H433K and N434F is shown as SEQ ID NO:17 in Table 1.

[0170] In some embodiments, a suitable Fc domain may contain mutations of L234A (Leu234Ala), L235A (Leu235Ala), H433K (His433Lys) and/or N434F (Asn434Phe) (EU numbering). As a non-limiting example, a suitable Fc domain may contain mutations L234A, L235A, H433K and N434F (EU numbering). In some embodiments, a suitable Fc domain may comprise mutations L234A/L235A, T350V, T366L, K392L, T394W. An exemplary Fe domain sequence incorporating the mutations L234A, L235A, H433K and N434F is shown as SEQ ID NO:18 in Table 1.

[0171] In some embodiments, Chain A and Chain B of a suitable Fc domain may contain sets of complementary mutations. These sets include, e.g., (i) Chain A: T350V/L351Y/F405A/Y407V. Chain B: T350V/T366L/K392LT394W; (ii); Chain A: T366W, S354C; Chain B: T366S, L368A, Y407V, Y349C; (iii) Chain A: K392D, K409D; Chain B: D399K, E356K: (iv) Chain A: K360E, K409W, Y349C; Chain B: Q347R, D399V, F405T, S354C; (v) Chain A: T366W; Chain B: T366S, L368A, Y407V; (vi) Chain A: K360E, K409W, Chain B: Q347R, D399V, F405T; and (vii) Chain A: T350V/L351Y/F405A/Y407V, Chain B: T350V/T366L/K392L/T394W.

[0172] Additional amino acid substitutions that can be included in the Fc domain include those described in, e.g., U.S. Pat. Nos. 6,277,375; 8,012,476; and 8,163,881, T. S. Von Kreutenstein, et al. Improving biophysical properties of a bispecific antibody scaffold to aid developability: quality by molecular design mAbs, 5 (2013), pp. 646-654; and Ha J-H, Kim J-E and Kim Y-S (2016) Immunoglobulin Fc Heterodimer Platform Technology: From Design to Applications in Therapeutic Antibodies and Proteins. Front. Immunol. 7:394; which are incorporated herein by reference.

[0173] Exemplary HO-1 Fusion Proteins

[0174] In some embodiments, the recombinant heme oxygenase protein is fused to Fc at the N-terminus. In some embodiments, the recombinant heme oxygenase protein is fused to the C-terminus.

[0175] In some embodiments, the recombinant heme oxygenase protein comprises a multimer comprising at least one monomer comprising an Fc domain fused to a recombinant heme oxygenase protein domain.

[0176] In some embodiments, the recombinant heme oxygenase protein comprises a multimer comprising at least one monomer comprising an Fc domain not fused to a recombinant heme oxygenase protein domain.

[0177] In some embodiments, the recombinant heme oxygenase protein is a dimer, trimer, tetramer, pentamer, or larger aggregate.

[0178] In some embodiments, a recombinant heme oxygenase dimeric protein comprises one monomer comprising an Fc domain (typically a human Fc domain) fused to a recombinant heme oxygenase protein domain and another monomer comprising an Fc domain (typically a human Fc domain) not fused to a recombinant heme oxygenase protein domain. The Fc domain can be fused either to the N-terminus or the C-terminus of the recombinant heme oxygenase protein domain, optionally via a linker. The optional linker can contain multiple (e.g., 1-4) repeats of the amono acid sequence GGGGS. The heme oxygenase protein domain can be an enzymatically active fragment of a heme oxygenase protein. For example, enzymatically active fragments suitable for use with the invention may comprise an amino acid sequence with residues 1-261 or 1-226 of SEQ ID NO:1. In particular embodiments, an Fc domain is fused to the C-terminus of the heme oxygenase protein domain, typically without a linker.

[0179] In some embodiments, the Fc domain comprises one or more mutations to enhance half-life, reduce aggregation and/or alter effector function.

[0180] In some embodiments, the IgG1 Fc region comprises amino acid substitutions at positions 234, 235, 251, 252, 254, 255, 256, 308, 309, 311, 312, 314, 385, 386, 387, 389, 428, 433, 434, 435 and/or 436 (according to EU numbering).

[0181] In some embodiments, the Fc region comprises L234A and L235A amino acid substitutions.

[0182] In some embodiments, the Fc domain fused to the recombinant heme oxygenase protein domain and the Fc domain not fused to a recombinant heme oxygenase protein domain contain complementary mutations that aid heterodimer formation. The complementary sets of mutations located in the two Fc domains (referred to as Chain A and Chain B, respectively) can include, e.g., (i) Chain A: T350V/L351Y/F405A/Y407V, Chain B: T350V/T366L/K392L/T394W: (ii); Chain A: T366W, S354C; Chain B: T366S, L368A, Y407V, Y349C; (iii) Chain A: K392D, K409D: Chain B: D399K, E356K; (iv) Chain A: K360E, K409W, Y349C; Chain B: Q347R, D399V, F405T, S354C: (v) Chain A: T366W; Chain B: T366S, L368A, Y407V; (vi) Chain A: K360E, K409W, Chain B: Q347R, D399V, F405T; and (vii) Chain A: T350V/L351Y/F405A/Y407V, Chain B: T350V/T366L/K392L/T394W. Recombinant heterodimeric proteins monovalent for a heme oxygenase domain (typically an enzymatically active fragment comprising an amino acid sequence with residues 1-261 or 1-226 of SEQ ID NO:1), which include Fe domains with complementary sets of mutations (e.g., sets (i), (iv) or (vii) as listed) above display robust enzymatic activity and can be purified at high yield.

[0183] In other embodiments, the monomer comprising the Fc domain fused to the recombinant heme oxygenase protein domain and the monomer comprising the Fc domain not fused to a recombinant heme oxygenase protein domain are linked (e.g., via a linker comprising multiple (e.g., 6-10) repeats of the amino acid sequence GGGGS), thus forming a single chain recombinant heme oxygenase protein. In particular embodiments, the two linked Fc domains are fused to the C-terminus of the heme oxygenase protein domain. Typically, no linker is required to fuse the two linked Fc domains to the heme oxygenase protein domain. For greater stability, the two linked Fc domains (referred to as Chain A and Chain B, respectively) may include reverse charge mutations, e.g., Chain A: E357K; Chain B: K370D.

[0184] In another embodiment, a recombinant heme oxygenase dimeric protein comprises two monomers, each comprising an Fc domain fused to a recombinant heme oxygenase protein domain. In this embodiment, the two monomers may be identical, resulting in the formation of a heme oxygenase bivalent homodimer. The Fc domain can be fused either to the N-terminus or the C-terminus of the recombinant heme oxygenase protein domain, optionally via a linker. The optional linker can contain multiple (e.g., 1-4) repeats of the amono acid sequence GGGGS. The heme oxygenase protein domain can be an enzymatically active fragment of a heme oxygenase protein. For example, enzymatically active fragments suitable for use with the invention comprise an amino acid sequence with residues 1-261 or 1-226 of SEQ ID NO:1. In some embodiments, an Fc domain is fused to the C-terminus of the heme oxygenase protein domain (e.g., an enzymatically active fragments comprising the amino acid sequence with residues 1-226 of SEQ ID NO:1), typically without a linker the fragment. Fusing an Fc domain to the C-terminus of a heme oxygenase protein domain has been found to yield a recombinant heme oxygenase homodimeric protein with robust enzymatic activity that can be purified with a high yield.

[0185] In certain embodiments, an engineered protein as described herein can be PEGylated to include mono- or poly-(e.g., 2-4) PEG moieties. Such PEGylated proteins may display increased half-life in comparison to a non-PEGylated reference protein, e.g., a protein having the same amino acid sequence but different, a different amount of, or no PEGylation. Methods for preparing a PEGylated protein can generally include (a) reacting a polypeptide with polyethylene glycol (such as a reactive ester or aldehyde derivative of PEG) under conditions whereby the polypeptide becomes attached to one or more PEG groups; and (b) obtaining the reaction product(s). In general, the conditions for the reactions can be determined case by case based on known parameters and the desired result. A number of PEG attachment methods are available to those skilled in the art. For example, the step of PEGylating a multi-specific binding molecule described herein can be carried out via an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule.

[0186] In some embodiments, serum half-life is increased by binding to a homo amino acid polymer (HAPylation), a proline-alanine-serine polymer (PAS, PASylation, or an elastin-like peptide (ELPylation), or fusion with artificial GLK.

[0187] In some embodiments, serum half-life of an engineered protein is increased. For example, binding of an engineered protein to FcRn increases serum half-life of the antibody to about 4 days to about 45 days, e.g., about 5 days to about 30 days, about 10 days to about 30 days, or about 20 days to about 30 days. In certain embodiments, an engineered antibody described herein has a serum half-life of about 5 days, about 10 days, about 15 days, about 20 days, about 25 days, about 30 days, about 35 days, about 40 days, about 45 days, about 50 days or longer.

[0188] In some embodiments, serum half-life is increased by binding to human serum albumin protein or transferrin protein or human IgG, via genetic fusion, or by non-covalent binding or chemical conjugation.

[0189] In some embodiments, serum half-life is increased by fusion with an anionic highly-sialylated peptide, such as the carboxy-terminal peptide (CTP, of chorionic gonadotropin β chain).

[0190] In some embodiments, serum half-life is increased by the use of a linker.

[0191] In some embodiments, aggregation is reduced by the use of a linker.

[0192] In some embodiments, the recombinant heme oxygenase protein comprises a linker sequence of GGGGS (SEQ ID NO: 10).

[0193] In some embodiments, aggregation is reduced by use of a monomer or single-chain moiety.

[0194] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 4 (1-261-Fc V1):

TABLE-US-00005 (SEQ ID NO: 4) MERPQPDSMPQDLSEALKEATKEVHTQAENAEFMRNFQKGQVTRDGFKLV MASLYHIYVALEEEIERNKESPVFAPVYFPEELHRKAALEQDLAFWYGPR WQEVIPYTPAMQRYVKRLHEVGRTEPELLVAHAYTRYLGDLSGGQVLKKI AQKALDLPSSGEGLAFFTFPNIASATKFKQLYRSRMNSLEMTPAVRQRVI EEAKTAFLLNIQLFEELQELLTHDTKDQSPSRAPGLRQRASNKVQDSAPV ETPRGKPPLNTDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0195] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising 1-161 Fc of SEQ ID NO: 4 and a signal peptide comprising MDMRVPAQLL GLLLLWFPGS RC (SEQ ID NO: 9).

[0196] In some embodiments, recombinant HO-1 comprises amino acid 1-261 of wild-type HO-1. In some embodiments, rHO-1 Fc fusions comprise an IgG1 Fc with LALA mutation. See Hezareh et al., J. Virol. 75, 12161-12168 (2001). In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 4.

[0197] In some embodiments, recombinant heme oxygenase comprises an Fc fusion comprising SEQ ID NO: 5 (1-261-Fc V2). In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 5.

TABLE-US-00006 (SEQ ID NO: 5) MERPQPDSMPQDLSEALKEATKEVHTQAENAEFMRNFQKGQVTRDGFKLV MASLYHIYVALEEEIERNKESPVFAPVYFPEELHRKAALEQDLAFWYGPR WQEVIPYTPAMQRYVKRLHEVGRTEPELLVAHAYTRYLGDLSGGQVLKKI AQKALDLPSSGEGLAFFTFPNIASATKFKQLYRSRMNSLEMTPAVRQRVI EEAKTAFLLNIQLFEELQELLTHDTKDQSPSRAPGLRQRASNKVQDSAPV ETPRGKPPLNT   DKTHTCPPCPAPEAAG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK

[0198] In some embodiments, rHO-1 comprises a signal peptide. In some embodiments, recombinant heme oxygenase is an Fc fusion comprising 1-161 Fc of SEQ ID NO: 5 and a signal peptide comprising MDMRVPAQLL GLLLLWFPGS RC (SEQ ID NO: 9).

[0199] In some embodiments, rHO-1 comprises a linker peptide. In some embodiments, the linker comprises GGGGS (SEQ ID NO: 10). In some embodiments, the linker comprises two or more repeats of GGGGS (SEQ ID NO: 62: “GGGGS” disclosed as SEQ ID NO: 10). In some embodiments, the linker comprises 4 repeats of GGGGS (SEQ ID NO: 21; “GGGGS” disclosed as SEQ ID NO: 10).

[0200] In some embodiments, recombinant heme oxygenase comprises an Fc fusion comprising SEQ ID NO: 6 (1-261-Fc V3). In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 6.

TABLE-US-00007 (SEQ ID NO: 6) MDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK ERPQPDSMPQDLSEALKEATKEVHTQAENAEFMRNFQKGQVTRDGFKLVM ASLYHIYVALEEEIERNKESPVFAPVYFPEELHRKAALEQDLAFWYGPRW QEVIPYTPAMQRYVKRLHEVGRTEPELLVAHAYTRYLGDLSGGQVLKKIA QKALDLPSSGEGLAFFTFPNIASATKFKQLYRSRMNSLEMTPAVRQRVIE EAKTAFLLNIQLFEELQELLTHDTKDQSPSRAPGLRQRASNKVQDSAPVE TPRGKPPLNT

[0201] In some embodiments, rHO-1 comprises a signal peptide. In some embodiments, the signal peptide comprises MDMRVPAQLL GLLLLWFPGS RC (SEQ ID NO: 9). In some embodiments, recombinant heme oxygenase is an Fc fusion comprising 1-161 Fc of SEQ ID NO: 6 and a signal peptide comprising MDMRVPAQLL GLLLLWFPGS RC (SEQ ID NO: 9).

[0202] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 7 (1-261-Fc V4 Chain A). In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 7.

TABLE-US-00008 (SEQ ID NO: 7) MERPQPDSMPQDLSEALKEATKEVHTQAENAEFMRNFQKGQVTRDGFKLV MASLYHIYVALEEEIERNKESPVFAPVYFPEELHRKAALEQDLAFWYGPR WQEVIPYTPAMQRYVKRLHEVGRTEPELLVAHAYTRYLGDLSGGQVLKKI AQKALDLPSSGEGLAFFTFPNIASATKFKQLYRSRMNSLEMTPAVRQRVI EEAKTAFLLNIQLFEELQELLTHDTKDQSPSRAPGLRQRASNKVQDSAPV ETPRGKPPLNTDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0203] In some embodiments, rHO-1 comprises a signal peptide. In some embodiments, recombinant heme oxygenase is an Fc fusion comprising 1-161 Fc of SEQ ID NO: 7 and a signal peptide comprising MDMRVPAQLL GLLLLWFPGS RC (SEQ ID NO: 9).

[0204] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 7 (1-261-Fc V4 Chain A). In some embodiments, the rHO-1 Fc fusion is expressed with a signal peptide (SEQ ID NO: 41). In some embodiments, the signal peptide is cleaved. In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 41.

TABLE-US-00009 (SEQ ID NO: 41) METPAQLLFLLLLWLPDTTGMERPQPDSMPQDLSEALKEATKEVHTQAEN AEFMRNFQKGQVTRDGFKLVMASLYHIYVALEEEIERNKESPVFAPVYFP EELHRKAALEQDLAFWYGPRWQEVIPYTPAMQRYVKRLHEVGRTEPELLV AHAYTRYLGDLSGGQVLKKIAQKALDLPSSGEGLAFFTFPNIASATKFKQ LYRSRMNSLEMTPAVRQRVIEEAKTAFLLNIQLFEELQELLTHDTKDQSP SRAPGLRQRASNKVQDSAPVETPRGKPPLNTDKTHTCPPCPAPEAAGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK

[0205] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 8 (1-261-Fc V4 Chain B). In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 8.

TABLE-US-00010 (SEQ ID NO: 8) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVK GFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK

[0206] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 8 (1-261-Fc V4 Chain B). In some embodiments, the rHO-1 Fc fusion is expressed with a signal peptide (SEQ ID NO: 42). In some embodiments, the signal peptide is cleaved. In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 42 including the signal peptide shown in bold below.

TABLE-US-00011 (SEQ ID NO: 42) METPAQLLFLLLLWLPDTTGDKTHTCPPCPAPEAAGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYV LPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0207] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 22 (1-261-Fc V5 Chain A, T366W and S354C: HO-1 underlined). In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 22.

TABLE-US-00012 (SEQ ID NO: 22) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGMERPQPDSMPQDLSEALKEATKEV HTQAENAEFMRNFQKGQVTRDGFKLVMASLYHIYVALEEEIERNKESPVF APVYFPEELHRKAALEQDLAFWYGPRWQEVIPYTPAMQRYVKRLHEVGRT EPELLVAHAYTRYLGDLSGGQVLKKIAQKALDLPSSGEGLAFFTFPNIAS ATKFKQLYRSRMNSLEMTPAVRQRVIEEAKTAFLLNIQLFEELQELLTHD TKDQSPSRAPGLRQRASNKVQDSAPVETPRGKPPLNT

[0208] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 22 (1-261-Fc V5 Chain A, T366W and S354C; HO-1 underlined). In some embodiments, the rHO-1 Fc fusion is expressed with a signal peptide (SEQ ID NO: 43). In some embodiments, the signal peptide is cleaved. In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 43 including the signal peptide shown in bold below.

TABLE-US-00013 (SEQ ID NO: 43) MGWSCHILFLVATATGVHSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGMERPQ PDSMPQDLSEALKEATKEVHTQAENAEFMRNFQKGQVTRDGFKLVMASLY HIYVALEEEIERNKESPVFAPVYFPEELHRKAALEQDLAFWYGPRWQEVI PYTPAMQRYVKRLHEVGRTEPELLVAHAYTRYLGDLSGGQVLKKIAQKAL DLPSSGEGLAFFTFPNIASATKFKQLYRSRMNSLEMTPAVRQRVIEEAKT AFLLNIQLFEELQELLTHDTKDQSPSRAPGLRQRASNKVQDSAPVETPRG KPPLNT

[0209] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 23 (1-261-Fc V5 Chain B. T366S, L368A, Y407V and Y349C). In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 23.

TABLE-US-00014 (SEQ ID NO: 23) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG

[0210] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 23 (1-261-Fc V5 Chain B, T366S, L368A, Y407V and Y349C). In some embodiments, the rHO-1 Fc fusion is expressed with a signal peptide (SEQ ID NO: 44). In some embodiments, the signal peptide is cleaved. In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 44 including the signal peptide shown in bold below.

TABLE-US-00015 (SEQ ID NO: 44) MGWSCHILFLVATATGVHSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTL PPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0211] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 24 (1-261-Fe V6 Chain A, K392D, K409D, bold). In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 24.

TABLE-US-00016 (SEQ ID NO: 24) SDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGMERPQPDSMPQDLSEALKEATKE VHTQAENAEFMRNFQKGQVTRDGFKLVMASLYHIYVALEEEIERNKESPV FAPVYFPEELHRKAALEQDLAFWYGPRWQEVIPYTPAMQRYVKRLHEVGR TEPELLVAHAYTRYLGDLSGGQVLKKIAQKALDLPSSGEGLAFFTFPNIA SATKFKQLYRSRMNSLEMTPAVRQRVIEEAKTAFLLNIQLFEELQELLTH DTKDQSPSRAPGLRQRASNKVQDSAPVETPRGKPPLNT

[0212] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 24 (1-261-Fc V6 Chain A, K392D, K409D). In some embodiments, the rHO-1 Fc fusion is expressed with a signal peptide (SEQ ID NO: 45). In some embodiments, the signal peptide is cleaved. In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 45 including the signal peptide shown in bold below.

TABLE-US-00017 (SEQ ID NO: 45) MGWSCHILFLVATATGVHSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSD GSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGMERPQ PDSMPQDLSEALKEATKEVHTQAENAEFMRNFQKGQVTRDGFKLVMASLY HIYVALEEEIERNKESPVFAPVYIPEEIHRKAALEQDLAFWYGPRWQEVI PYTPAMQRYVKRLHEVGRTEPELLVAHAYTRYLGDLSGGQVLKKIAQKAL DLPSSGEGLAFFTFPNIASATKFKQLYRSRMNSLEMTPAVRQRVIEEAKT AFLLNIQLFEELQELLTHDTKDQSPSRAPGLRQRASNKVQDSAPVETPRG KPPLNT

[0213] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 25 (1-261-Fc V6 Chain B, D399K. E356K, bold). In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 25.

TABLE-US-00018 (SEQ ID NO: 25) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG

[0214] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 25 (1-261-Fc V6 Chain B, D399K, E356K). In some embodiments, the rHO-1 Fc fusion is expressed with a signal peptide (SEQ ID NO: 46). In some embodiments, the signal peptide is cleaved. In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 46 including the signal peptide shown in bold below.

TABLE-US-00019 (SEQ ID NO: 46) MGWSCIILFLVATATGVHSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRKELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0215] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 26 (1-261-Fc V7 Chain A, K360E, K409W, Y349C, bold). In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 901%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 26.

TABLE-US-00020 (SEQ ID NO: 26) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTENQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSWLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGMERPQPDSMPQDLSEALKEATKEV HTQAENAEFMRNFQKGQVTRDGFKLVMASLYHIYVALEEEIERNKESPVF APVYFPEELHRKAALEQDLAFWYGPRWQEVIPYTPAMQRYVKRLHEVGRT EPELLVAHAYTRYLGDLSGGQVLKKIAQKALDLPSSGEGLAFFTFPNIAS ATKFKQLYRSRMNSLEMTPAVRQRVIEEAKTAFLLNIQLFEELQELLTHD TKDQSPSRAPGLRQRASNKVQDSAPVETPRGKPPLNT

[0216] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 26 (1-261-Fc V7 Chain A, K360E, K409W, Y349C). In some embodiments, the rHO-1 Fc fusion is expressed with a signal peptide (SEQ ID NO: 47). In some embodiments, the signal peptide is cleaved. In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 47 including the signal peptide shown in bold below.

TABLE-US-00021 (SEQ ID NO: 47) MGWSCIILFLVATATGVHSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTL PPSRDELTENQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSWLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGMERPQ PDSMPQDLSEALKEATKEVHTQAENAEFMRNFQKGQVTRDGFKLVMASLY HIYVALEEEIERNKESPVFAPVYFPEELHRKAALEQDLAFWYGPRWQEVI PYTPAMQRYVKRLHEVGRTEPELLVAHAYTRYLGDLSGGQVLKKIAQKAL DLPSSGEGLAFFTFPNIASATKFKQLYRSRMNSLEMTPAVRQRVIEEAKT AFLLNIQLFEELQELLTHDTKDQSPSRAPGLRQRASNKVQDSAPVETPRG KPPLNT

[0217] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 27 (1-261-Fc V7 Chain B, Q347R, D399V, F405T, S354C, bold). In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 27.

TABLE-US-00022 (SEQ ID NO: 27) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPRVYTLPPCRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLVSDGSFTLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG

[0218] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 27 (1-261-Fc V7 Chain B. Q347R, D399V, F405T, S354C). In some embodiments, the rHO-1 Fc fusion is expressed with a signal peptide (SEQ ID NO: 48). In some embodiments, the signal peptide is cleaved. In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 48 including the signal peptide shown in bold below.

TABLE-US-00023 (SEQ ID NO: 48) MGWSCIILFLVATATGVHSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPRVYTL PPCRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLVSD GSFTLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0219] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 28 (1-261-Fc V8 Chain A, T366V, bold. HO-1 underlined). In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 28.

TABLE-US-00024 (SEQ ID NO: 28) MERPQPDSMPQDLSEALKEATKEVHTQAENAEFMRNFQKGQVTRDGFKLV MASLYHIYVALEEEIERNKESPVFAPVYFPEELHRKAALEQDLAFWYGPR WQEVIPYTPAMQRYVKRLHEVGRTEPELLVAHAYTRYLGDLSGGQVLKKI AQKALDLPSSGEGLAFFTFPNIASATKFKQLYRSRMNSLEMTPAVRQRVI EEAKTAFLLNIQLFEELQELLTHDTKDQSPSRAPGLRQRASNKVQDSAPV ETPRGKPPLNTDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0220] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 28 (1-261-Fc V8 Chain A, T366W). In some embodiments, the rHO-1 Fc fusion is expressed with a signal peptide (SEQ ID NO: 49). In some embodiments, the signal peptide is cleaved. In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 49 including the signal peptide shown in bold below.

TABLE-US-00025 (SEQ ID NO: 49) MGWSCHILFLVATATGVHSMERPQPDSMPQDLSEALKEATKEVHTQAENA EFMRNFQKGQVTRDGFKLVMASLYHIYVALEEEIERNKESPVFAPVYFPE ELHRKAALEQDLAFWYGPRWQEVIPYTPAMQRYVKRLHEVGRTEPELLVA HAYTRYLGDLSGGQVLKKIAQKALDLPSSGEGLAFFTFPNIASATKFKQL YRSRMNSLEMTPAVRQRVIEEAKTAFLLNIQLFEELQELLTHDTKDQSPS RAPGLRQRASNKVQDSAPVETPRGKPPLNTDKTHTCPPCPAPEAAGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG

[0221] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 29 (1-261-Fc V8 Chain B, T366S, L368A, Y407V, bold). In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 29.

TABLE-US-00026 (SEQ ID NO: 29) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG

[0222] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 29 (1-261-Fc V8 Chain B, T366S, L368A, Y407V). In some embodiments, the rHO-1 Fc fusion is expressed with a signal peptide (SEQ ID NO: 50). In some embodiments, the signal peptide is cleaved. In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 50 including the signal peptide shown in bold below.

TABLE-US-00027 (SEQ ID NO: 50) MGWSCIILFLVATATGVHSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0223] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 30 (1-261-Fc V9 Chain A, K360E, K409W, bold). In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 30.

TABLE-US-00028 (SEQ ID NO: 30) MERPQPDSMPQDLSEALKEATKEVHTQAENAEFMRNFQKGQVTRDGFKLV MASLYHIYVALEEEIERNKESPVFAPVYFPEELHRKAALEQDLAFWYGPR WQEVIPYTPAMQRYVKRLHEVGRTEPELLVAHAYTRYLGDLSGGQVLKKI AQKALDLPSSGEGLAFFTFPNIASATKFKQLYRSRMNSLEMTPAVRQRVI EEAKTAFLLNIQLFEELQELLTHDTKDQSPSRAPGLRQRASNKVQDSAPV ETPRGKPPLNTDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT ENQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSW LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0224] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 30 (1-261-Fc V9 Chain A, K360E, K409W). In some embodiments, the rHO-1 Fc fusion is expressed with a signal peptide (SEQ ID NO: 51). In some embodiments, the signal peptide is cleaved. In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 51 including the signal peptide shown in bold below.

TABLE-US-00029 (SEQ ID NO: 51) MGWSCIILFLVATATGVHSMERPQPDSMPQDLSEALKEATKEVHTQAENA EFMRNFQKGQVTRDGFKLVMASLYHIYVALEEEIERNKESPVFAPVYFPE ELHRKAALEQDLAFWYGPRWQEVIPYTPAMQRYVKRLHEVGRTEPELLVA HAYTRYLGDLSGGQVLKKIAQKALDLPSSGEGLAFFTFPNIASATKFKQL YRSRMNSLEMTPAVRQRVIEEAKTAFLLNIQLFEELQELLTHDTKDQSPS RAPGLRQRASNKVQDSAPVETPRGKPPLNTDKTHTCPPCPAPEAAGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTENQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSWLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG

[0225] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 31 (1-261-Fc V9 Chain B, Q347R, D399V, F405T, bold). In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 31.

TABLE-US-00030 (SEQ ID NO: 31) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPRVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLVSDGSFTLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG

[0226] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 31 (1-261-Fc V9 Chain B, Q347R, D399V, F405T). In some embodiments, the rHO-1 Fc fusion is expressed with a signal peptide (SEQ ID NO: 52). In some embodiments, the signal peptide is cleaved. In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 52 including the signal peptide shown in bold below.

TABLE-US-00031 (SEQ ID NO: 52) MGWSCHILFLVATATGVHSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPRVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLVSD GSFTLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0227] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 32 (1-226-Fc V10 Chain A, T350V, L351Y, F405A, Y407V, bold; HO-1 underlined). In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 31.

TABLE-US-00032 (SEQ ID NO: 32) MERPQPDSMPQDLSEALKEATKEVHTQAENAEFMRNFQKGQVTRDGFKLV MASLYHIYVALEEEIERNKESPVFAPVYFPEELHRKAALEQDLAFWYGPR WQEVIPYTPAMQRYVKRLHEVGRTEPELLVAHAYTRYLGDLSGGQVLKKI AQKALDLPSSGEGLAFFTFPNIASATKFKQLYRSRMNSLEMTPAVRQRVI EEAKTAFLLNIQLFEELQELLTHDTKDKTHTCPPCPAPEAAGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PG

[0228] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 32 (1-226-Fc V10 Chain A, T350V, L351Y, F405A, Y407V). In some embodiments, the rHO-1 Fc fusion is expressed with a signal peptide (SEQ ID NO: 53). In some embodiments, the signal peptide is cleaved. In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 53 including the signal peptide shown in bold below.

TABLE-US-00033 (SEQ ID NO: 53) METPAQLLFLLLLWLPDTTGMERPQPDSMPQDLSEALKEATKEVHTQAEN AEFMRNFQKGQVTRDGFKLVMASLYHIYVALEEEIERNKESPVFAPVYFP EELHRKAALEQDLAFWYGPRWQEVIPYTPAMQRYVKRLHEVGRTEPELLV AHAYTRYLGDLSGGQVLKKIAQKALDLPSSGEGLAFFTFPNIASATKFKQ LYRSRMNSLEMTPAVRQRVIEEAKTAFLLNIQLFEELQELLTHDTKDKTH TCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPG

[0229] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 33 (1-226-Fc V10 Chain B, T350V, T366L, K392L, T394W, bold). In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 33.

TABLE-US-00034 (SEQ ID NO: 33) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLLC LVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0230] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 33 (1-226-Fc V10 Chain B, T350V, T366L, K392L, T394W). In some embodiments, the rHO-1 Fc fusion is expressed with a signal peptide (SEQ ID NO: 54). In some embodiments, the signal peptide is cleaved. In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 900%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 54 including the signal peptide shown in bold below.

TABLE-US-00035 (SEQ ID NO: 54) METPAQLLFLLLLWLPDTTGDKTHTCPPCPAPEAAGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G

[0231] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 34 (1-226-Fc V11; HO-1 underlined). In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 34.

TABLE-US-00036 (SEQ ID NO: 34) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGMERPQPDSMPADLSEALKE ATKEVHTAAENAEFMRNFQKGAVTRDGFKLVMASLYHIYVALEEEIERN KESPVFAPVYFPEELHRKAALEADLAFWYGPRWAEVIPYTPAMARYVKR LHEVGRTEPELLVAHAYTRYLGDLSGGAVLKKIAAKALDLPSSGEGLAF FTFPNIASATKFKALYRSRMNSLEMTPAVRARVIEEAKTAFLLNIALFE ELAELLTHDTK

[0232] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 34 (1-226-Fc V11). In some embodiments, the rHO-1 Fc fusion is expressed with a signal peptide (SEQ ID NO: 55). In some embodiments, the signal peptide is cleaved. In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 55 including the signal peptide shown in bold below.

TABLE-US-00037 (SEQ ID NO: 55) MGWSCHILFLVATATGVHSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG MERPQPDSMPQDLSEALKEATKEVHTQAENAEFMRNFQKGQVTRDGFKL VMASLYHIYVALEEEIERNKESPVFAPVYFPEELHRKAALEQDLAFWYG PRWQEVIPYTPAMQRYVKRLHEVGRTEPELLVAHAYTRYLGDLSGGQVL KKIAQKALDLPSSGEGLAFFTFPNIASATKFKQLYRSRMNSLEMTPAVR QRVIEEAKTAFLLNIQLFEELQELLTHDTK

[0233] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 35 (1-226-Fc V12: HO-1 underlined). In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 35.

TABLE-US-00038 (SEQ ID NO: 35) MGWSCHILFLVATATGVHSMERPQPDSMPQDLSEALKEATKEVHTQAEN AEFMRNFQKGQVTRDGFKLVMASLYHIYVALEEEIERNKESPVFAPVYF PEELHRKAALEQDLAFWYGPRWQEVIPYTPAMQRYVKRLHEVGRTEPEL LVAHAYTRYLGDLSGGQVLKKIAQKALDLPSSGEGLAFFTFPNIASATK FKQLYRSRMNSLEMTPAVRQRVIEEAKTAFLLNIQLFEELQELLTHDTK DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0234] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 35 (1-226-Fc V12). In some embodiments, the rHO-1 Fc fusion is expressed with a signal peptide (SEQ ID NO: 56). In some embodiments, the signal peptide is cleaved. In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 56 including the signal peptide shown in bold below.

TABLE-US-00039 (SEQ ID NO: 56) MGWSCIILFLVATATGVHSMERPQPDSMPQDLSEALKEATKEVHTQAEN AEFMRNFQKGQVTRDGFKLVMASLYHIYVALEEEIERNKESPVFAPVYF PEELHRKAALEQDLAFWYGPRWQEVIPYTPAMQRYVKRLHEVGRTEPEL LVAHAYTRYLGDLSGGQVLKKIAQKALDLPSSGEGLAFFTFPNIASATK FKQLYRSRMNSLEMTPAVRQRVIEEAKTAFLLNIQLFEELQELLTHDTK DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0235] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 36 (1-261-Fc V13; HO-1 underlined). In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 36.

TABLE-US-00040 (SEQ ID NO: 36) MERPQPDSMPQDLSEALKEATKEVHTQAENAEFMRNFQKGQVTRDGFKL VMASLYHIYVALEEEIERNKESPVFAPVYFPEELHRKAALEQDLAFWYG PRWQEVIPYTPAMQRYVKRLHEVGRTEPELLVAHAYTRYLGDLSGGQVL KKIAQKALDLPSSGEGLAFFTFPNIASATKFKQLYRSRMNSLEMTPAVR QRVIEEAKTAFLLNIQLFEELQELLTHDTKDQSPSRAPGLRQRASNKVQ DSAPVETPRGKPPLNTDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGG GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGEPKSSDKTHTC PPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG

[0236] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 36 (1-261-Fc V13). In some embodiments, the rHO-1 Fc fusion is expressed with a signal peptide (SEQ ID NO: 57). In some embodiments, the signal peptide is cleaved. In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 57 including the signal peptide shown in bold below.

TABLE-US-00041 (SEQ ID NO: 57) MGWSCIILFLVATATGVHSMERPQPDSMPQDLSEALKEATKEVHTQAEN AEFMRNFQKGQVTRDGFKLVMASLYHIYVALEEEIERNKESPVFAPVYF PEELHRKAALEQDLAFWYGPRWQEVIPYTPAMQRYVKRLHEVGRTEPEL LVAHAYTRYLGDLSGGQVLKKIAQKALDLPSSGEGLAFFTFPNIASATK FKQLYRSRMNSLEMTPAVRQRVIEEAKTAFLLNIQLFEELQELLTHDTK DQSPSRAPGLRQRASNKVQDSAPVETPRGKPPLNTDKTHTCPPCPAPEA AGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGG GSGGGGSGEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVIHQDW1NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0237] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 37 (1-261-Fc V14; HO-1 underlined). In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 37.

TABLE-US-00042 (SEQ ID NO: 37) MERPQPDSMPQDLSEALKEATKEVHTQAENAEFMRNFQKGQVTRDGFKL VMASLYHIYVALEEEIERNKESPVFAPVYFPEELHRKAALEQDLAFWYG PRWQEVIPYTPAMQRYVKRLHEVGRTEPELLVAHAYTRYLGDLSGGQVL KKIAQKALDLPSSGEGLAFFTFPNIASATKFKQLYRSRMNSLEMTPAVR QRVIEEAKTAFLLNIQLFEELQELLTHDTKDQSPSRAPGLRQRASNKVQ DSAPVETPRGKPPLNTPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDKLTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGG GGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSG GGGSPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVDGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPG

[0238] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 37 (1-261-Fc V14). In some embodiments, the rHO-1 Fc fusion is expressed with a signal peptide (SEQ ID NO: 58). In some embodiments, the signal peptide is cleaved. In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 58 including the signal peptide shown in bold below.

TABLE-US-00043 (SEQ ID NO: 58) MGWSCIILFLVATATGVHSMERPQPDSMPQDLSEALKEATKEVHTQAEN AEFMRNFQKGQVTRDGFKLVMASLYHIYVALEEEIERNKESPVFAPVYF PEELHRKAALEQDLAFWYGPRWQEVIPYTPAMQRYVKRLHEVGRTEPEL LVAHAYTRYLGDLSGGQVLKKIAQKALDLPSSGEGLAFFTFPNIASATK FKQLYRSRMNSLEMTPAVRQRVIEEAKTAFLLNIQLFEELQELLTHDTK DQSPSRAPGLRQRASNKVQDSAPVETPRGKPPLNTPAPEAAGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDKLTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGG GGSGGGGSGGGGSGGGGSGGGGSPAPEAAGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0239] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 38 (1-226-Fc V15: HO-1 underlined). In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 38.

TABLE-US-00044 (SEQ ID NO: 38) MERPQPDSMPQDLSEALKEATKEVHTQAENAEFMRNFQKGQVTRDGFKL VMASLYHIYVALEEEIERNKESPVFAPVYFPEELHRKAALEQDLAFWYG PRWQEVIPYTPAMQRYVKRLHEVGRTEPELLVAHAYTRYLGDLSGGQVL KKIAQKALDLPSSGEGLAFFTFPNIASATKFKQLYRSRMNSLEMTPAVR QRVIEEAKTAFLLNIQLFEELQELLTHDTKDKTHTCPPCPAPEAAGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGG GSGEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVITVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0240] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 38 (1-226-Fc V15). In some embodiments, the rHO-1 Fc fusion is expressed with a signal peptide (SEQ ID NO: 59). In some embodiments, the signal peptide is cleaved. In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 59 including the signal peptide shown in bold below.

TABLE-US-00045 (SEQ ID NO: 59) MGWSCHILFLVATATGVHSMERPQPDSMPQDLSEALKEATKEVHTQAEN AEFMRNFQKGQVTRDGFKLVMASLYHIYVALEEEIERNKESPVFAPVYF PEELHRKAALEQDLAFWYGPRWQEVIPYTPAMQRYVKRLHEVGRTEPEL LVAHAYTRYLGDLSGGQVLKKIAQKALDLPSSGEGLAFFTFPNIASATK FKQLYRSRMNSLEMTPAVRQRVIEEAKTAFLLNIQLFEELQELLTHDTK DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGG SGGGGSGGGGSGGGGSGGGGSGEPKSSDKTHTCPPCPAPEAAGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPG

[0241] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO: 39 (1-226-Fc V16; HO-1 underlined). In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 39.

TABLE-US-00046 (SEQ ID NO: 39) MERPQPDSMPQDLSEALKEATKEVHTQAENAEFMRNFQKGQVTRDGFKL VMASLYHIYVALEEEIERNKESPVFAPVYFPEELHRKAALEQDLAFWYG PRWQEVIPYTPAMQRYVKRLHEVGRTEPELLVAHAYTRYLGDLSGGQVL KKIAQKALDLPSSGEGLAFFTFPNIASATKFKQLYRSRMNSLEMTPAVR QRVIEEAKTAFLLNIQLFEELQELLTHDTKPAPPAAGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSRDKLTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGG GGSGGGGSGGGGSGGGGSPAPEAAGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVDGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 

[0242] In some embodiments, recombinant heme oxygenase is an Fc fusion comprising SEQ ID NO:39 (1-226-Fc V16). In some embodiments, the rHO-1 Fc fusion is expressed with a signal peptide (SEQ ID NO: 60). In some embodiments, the signal peptide is cleaved. In some embodiments, rHO-1-Fc fusion comprises a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 60 including the signal peptide shown in bold below.

TABLE-US-00047 (SEQ ID NO: 60) MGWSCHILFLVATATGVHSMERPQPDSMPQDLSEALKEATKEVHTQAEN AEFMRNFQKGQVTRDGFKLVMASLYHIYVALEEEIERNKESPVFAPVYF PEELHRKAALEQDLAFWYGPRWQEVIPYTPAMQRYVKRLHEVGRTEPEL LVAHAYTRYLGDLSGGQVLKKIAQKALDLPSSGEGLAFFTFPNIASATK FKQLYRSRMNSLEMTPAVRQRVIEEAKTAFLLNIQLFEELQELLTHDTK PAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDKLTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSPAPEAAGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVDGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG

[0243] Linker or Spacer

[0244] An HO-1 domain may be directly or indirectly linked to an Fc domain. In some embodiments, a suitable recombinant HO-1 protein comprises a linker or spacer that joins a HO-1 domain and an Fc domain. An amino acid linker or spacer is generally designed to be flexible or to interpose a structure, such as an alpha-helix, between the two protein moieties. A linker or spacer can be relatively short, or can be longer. Typically, a linker or spacer comprises for example 3-100 (e.g., 5-100, 10-100, 20-100 30-100, 40-100, 50-100, 60-100, 70-100, 80-100, 90-100, 5-55, 10-50, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20) amino acids in length. In some embodiments, a linker or spacer is equal to or longer than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length. Typically, a longer linker may decrease steric hindrance. In some embodiments, a linker will comprise a mixture of glycine and serine residues. In some embodiments, the linker may additionally comprise threonine, proline and/or alanine residues. Thus, in some embodiments, the linker comprises between 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 1040, 10-30, 10-20, 10-15 amino acids. In some embodiments, the linker comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 amino acids.

[0245] As non-limiting examples, linkers or spacers suitable for the present invention include but are not limited to:

TABLE-US-00048 (SEQ ID NO: 10) GGGGS (SEQ ID NO: 10) (GGGGS)n (SEQ ID NO: 11) GGG; (GAG linker, SEQ ID NO: 12) GAPGGGGGAAAAAGGGGGGAP; (GAG2 linker, SEQ ID NO:13) GAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAP; (GAG3 linker, SEQ ID NQ: 14) GAPGGGGGAAAAAGGGGGGAPGGGGGAAAAAGGGGGGAPGGGGGAAAA AGGGGGGAP; and (SEQ ID NQ: 65) GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS.

[0246] Suitable linkers or spacers also include those having an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous or identical to the above exemplary linkers. e.g., GAG linker (SEQ ID NO:12). GAG2 linker (SEQ ID NO:13), or GAG3 linker (SEQ ID NO:14). Additional linkers suitable for use with some embodiments may be found in US20120232021, filed on Mar. 2, 2012, the disclosure of which is hereby incorporated by reference in its entirety.

[0247] In some embodiments, a linker is provided that associates the HO-1 polypeptide with the Fc domain without substantially affecting the ability of the HO-1 polypeptide to bind to any of its cognate ligands (e.g., heme). In some embodiments, a linker is provided such that the binding of a HO-1 peptide to heme is not altered as compared to the HO-1 polypeptide alone.

Nucleotide Sequences

[0248] The present disclosure includes nucleotide sequences encoding one or more signal peptides, light chains, heavy chains, heavy chain constant domains, light chain constant domains, or other immunoglobulin-like sequences, antibodies, linker sequences, sequences comprising heme oxygenase proteins or fragments thereof, including variants, or binding molecules disclosed herein. In various instances, such nucleotide sequences may be present in a vector. In various instances such nucleotides may be present in the genome of a cell, e.g., a cell of a subject in need of treatment or a cell for production of a truncated protein or a fusion protein, e.g. a mammalian cell for production of a truncated protein or a fusion protein. In some embodiments, the nucleotide sequences for protein expression of HO-1 (e.g., truncated HO-1 and HO-1 Fc fusion proteins) are codon optimized. In some embodiments, the nucleotide sequences are codon optimized for protein expression to facilitate E. coli expression of HO-1. In some embodiments, the nucleotide sequences are codon optimized for protein expression to facilitate CHO cell expression of HO-1 Fc fusion proteins (e.g., 1-261-Fc V1-V4).

[0249] The term “Fc fragment”, as used herein, refers to one or more fragments of an Fc region that retains an Fc function and/or activity described herein, such as binding to an Fc receptor.

[0250] In some embodiments, a multi-specific molecule described herein is an engineered antibody (e.g., engineered to have pH sensitive binding to antigen and to FcRn).

[0251] In some embodiments, a binding moiety is or includes an antibody (e.g., an IgG antibody, e.g., an IgG1, IgG2, or IgG3 antibody), or an antigen binding fragment, engineered to bind to a target (i.e., antigen) in an altered manner (e.g., in a pH sensitive manner, e.g., in a more or less pH sensitive manner) relative to a reference antibody or antigen binding fragment. For example, an antibody can be engineered by modifying (e.g., by adding, deleting, or substituting) an amino acid within one or more antibody CDRs and/or at a position involved in antibody CDR structure. Exemplary, non-limiting sites of an antibody that can be modified include the following (amino acid positions are indicated based on the Kabat numbering (Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH)).

[0252] Heavy chain: H27, H31, H32, H33, H35, H50, H58, H59, H61, H62, H63, H64, H65, H99, H100b, and H102

[0253] Light chain: L24, L27, L28, L32, L53, L54, L56, L90, L92, and L94.

[0254] In some embodiments, one or more of these disclosed amino acids can be substituted with histidine, arginine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, or glutamine. Without wishing to be bound by theory, it is believed that substituting an amino acid at one or more of these positions with a histidine can result in an antibody having pH-dependent antigen-binding properties. In some embodiments, a non-histidine residue is substituted with a histidine residue. In some embodiments, a histidine residue is substituted with a non-histidine residue. Additional engineered antigen binding regions include those described in, e.g., U.S. Publ. No. 20110229489.

[0255] In some instances, a binding moiety is or includes an antibody constant region, Fc region or Fc fragment that binds one or more Fc receptors (e.g., FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB, FcγRIV, or FcRn receptor). In some embodiments, a constant region, Fc region or Fc fragment is engineered to bind to a target (e.g., an Fc receptor) in an altered manner (e.g., in a pH sensitive manner, e.g., in a more or less pH sensitive manner) relative to a reference constant region, Fc region or Fc fragment.

[0256] In some instances, a binding moiety can be or include a constant region, Fc region or Fc fragment of an IgG antibody engineered to include an amino acid addition, deletion, or substitution, of one or more of amino acid residues described herein (e.g., 251-256, 285-290, 308-314, 385-389, and 428-436 (Kabat numbering (Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH))).

Recombinant Gene Technology

[0257] In accordance with the present disclosure, there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are described in the literature (see, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.: DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. (1985)); Transcription And Translation (B. D. Hames & S. J. Higgins, eds. (1984)); Animal Cell Culture (R. I. Freshney, ed. (1986)); Immobilized Cells and Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

[0258] Recombinant expression of a gene, such as a nucleic acid encoding a polypeptide, such as an engineered antibody described herein, can include construction of an expression vector containing a nucleic acid that encodes the polypeptide. Once a polynucleotide has been obtained, a vector for the production of the polypeptide can be produced by recombinant DNA technology using techniques known in the art. Known methods can be used to construct expression vectors containing polypeptide coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.

[0259] An expression vector can be transferred to a host cell by conventional techniques, and the transfected cells can then be cultured by conventional techniques to produce polypeptides.

Methods of Treatment

[0260] The present disclosure also provides a recombinant heme oxygenase-1 (rHO-1) of the invention for use in a method of treating sickle cell disease in a subject in need of such treatment. As described herein, the present disclosure provides a method of treating sickle cell disease comprising administering to a subject in need of treatment a recombinant heme oxygenase-1 (rHO-1). The present disclosure also provides a recombinant heme oxygenase-1 (rHO-1) of the invention for use in the manufacture of a medicament for treating sickle cell disease in a subject in need of such treatment.

[0261] In some embodiments, recombinant heme oxygenase protein is administered intravenously.

[0262] In some embodiments, administration of recombinant heme oxygenase to a subject results in a reduction of free heme level in serum by 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55% or 50% compared to a control sample. The control sample can be a serum sample taken from the subject prior to treatment with the recombinant heme oxygenase.

[0263] In some embodiments, administration of recombinant heme oxygenase results in reduced free heme level in serum. In some embodiments, free heme levels in serum are reduced by about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 2%. In some embodiments, free heme levels are reduced by 5 μM, 10 μM, 11, 12, 13, 14, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM or more to approach levels in healthy adults (e.g., about 20 μM).

[0264] In some embodiments, administration of recombinant heme oxygenase to a subject results in increased recombinant heme oxygenase activity by 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more in scrum relative to a control individual. In some embodiments, administration of recombinant heme oxygenase to a subject results in increased recombinant heme oxygenase activity by 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold, or more in serum relative to a control individual. The control individual can be the subject prior to administration of recombinant heme oxygenase.

[0265] In some embodiments, a control individual has endogenous HO-1 levels of approximately 1 ng/ml (See e.g., Bao, et al. PLoS, 2010).

[0266] In some embodiments, administration of recombinant heme oxygenase results in increased recombinant HO-1 activity in serum at or above 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more relative to normal serum HO-1 activity in a healthy individual.

[0267] In some embodiments, administration of recombinant heme oxygenase to a subject in need of treatment results in HO-1 activity in serum at or above 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% of normal serum HO-1 activity in a healthy individual.

[0268] In some embodiments, administration of recombinant heme oxygenase results in amelioration of symptoms of sickle cell disease, including reduced or delayed onset of anemia, vasoocclusive crises (VOC), acute chest syndrome (ACS), pulmonary hypertension or organ damage.

[0269] In some embodiments, administration of recombinant heme oxygenase results in peripheral blood oxygen saturation values above 90%, 92%, 94%, 96%, 98%, or 99%.

[0270] In some embodiments, administration of recombinant heme oxygenase to a subject results in an increase in peripheral blood oxygen saturation values by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 95% compared to a control. The control can be peripheral blood oxygen saturation values taken from the subject prior to treatment with the recombinant heme oxygenase.

[0271] In some embodiments, administration of recombinant heme oxygenase results in 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% increase in breaths per minute, approaching 12-18 breaths per minute in normal healthy humans.

[0272] In some embodiments, administration of recombinant heme oxygenase results in 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% improvement in heart rate per minute, approaching 60-100 bpm in normal healthy humans.

Pharmaceutical Composition and Administration

[0273] The present invention further provides pharmaceutical compositions comprising therapeutically active ingredients in accordance with the invention (e.g., recombinant heme oxygenase protein, recombinant heme oxygenase fusion protein or recombinant heme oxygenase-Fc fusion protein), together with one or more pharmaceutically acceptable carriers or excipients. Such pharmaceutical compositions may optionally comprise one or more additional therapeutically-active substances.

[0274] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation.

[0275] Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a diluent or another excipient or carrier and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit.

[0276] A pharmaceutical composition in accordance with the invention may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[0277] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient or carrier, and/or any additional ingredients in a pharmaceutical composition in accordance with the invention will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.

[0278] Pharmaceutical formulations may additionally comprise a pharmaceutically acceptable excipient or carrier, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21.sup.st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporated herein by reference) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional excipient medium or carrier is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention.

[0279] In some embodiments, a pharmaceutically acceptable excipient or carrier is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient or carrier is approved for use in humans and for veterinary use. In some embodiments, an excipient or carrier is approved by United States Food and Drug Administration. In some embodiments, an excipient or carrier is pharmaceutical grade. In some embodiments, an excipient or carrier meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.

[0280] Pharmaceutically acceptable excipients or carriers used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients or carriers may optionally be included in pharmaceutical formulations. Excipients or carriers such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents can be present in the composition, according to the judgment of the formulator.

[0281] Suitable pharmaceutically acceptable excipients or carriers include but are not limited to water, salt solutions (e.g., NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, sugars such as mannitol, sucrose, or others, dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc., as well as combinations thereof. The pharmaceutical preparations can, if desired, be mixed with auxiliary agents (e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like) which do not deleteriously react with the active compounds or interfere with their activity. In a preferred embodiment, a water-soluble carrier suitable for intravenous administration is used.

[0282] A suitable pharmaceutical composition or medicament, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. A composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. A composition can also be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose, magnesium carbonate, etc.

[0283] A pharmaceutical composition or medicament can be formulated in accordance with the routine procedures as a pharmaceutical composition adapted for administration to human beings. For example, in some embodiments, a composition for intravenous administration typically is a solution in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0284] A recombinant heme oxygenase protein or recombinant heme oxygenase-Fc fusion protein described herein can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[0285] General considerations in the formulation and/or manufacture of pharmaceutical agents may be found, for example, in Remington: The Science and Practice of Pharmacy 21.sup.st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference).

Routes of Administration

[0286] A recombinant heme oxygenase protein or recombinant heme oxygenase-Fc fusion protein described herein (or a composition or medicament containing a recombinant heme oxygenase protein described herein) is administered by any appropriate route. In some embodiments, a recombinant heme oxygenase protein, recombinant heme oxygenase-Fc fusion protein or a pharmaceutical composition containing the same is administered systemically. Systemic administration may be intravenous, intradermal, inhalation, transdermal (topical), intraocular, intramuscular, subcutaneous, intramuscular, oral and/or transmucosal administration. In some embodiments, a recombinant heme oxygenase protein, recombinant heme oxygenase-Fc fusion protein or a pharmaceutical composition containing the same is administered subcutaneously. As used herein, the term “subcutaneous tissue”, is defined as a layer of loose, irregular connective tissue immediately beneath the skin. For example, the subcutaneous administration may be performed by injecting a composition into areas including, but not limited to, the thigh region, abdominal region, gluteal region, or scapular region. In some embodiments, a recombinant heme oxygenase protein, recombinant heme oxygenase-Fc fusion protein or a pharmaceutical composition comprising the same is administered intravenously. In some embodiments, a recombinant heme oxygenase protein, recombinant heme oxygenase-Fc fusion protein or a pharmaceutical composition containing the same is administered orally. In some embodiments, a recombinant heme oxygenase protein, recombinant heme oxygenase-Fc fusion protein or a pharmaceutical composition containing the same is administered intramuscularly. In some embodiments, more than one route can be used concurrently.

[0287] In some embodiments, administration results only in a localized effect in an individual, while in other embodiments, administration results in effects throughout multiple portions of an individual, for example, systemic effects. Typically, administration results in delivery of a recombinant heme oxygenase protein or recombinant heme oxygenase-Fc fusion protein systemically. In some embodiments, the recombinant heme oxygenase protein or recombinant heme oxygenase-Fc fusion protein is delivered to one or more target tissues including, but not limited to, heart, brain, spinal cord, striated muscle (e.g., skeletal muscle), smooth muscle, kidney, liver, lung, and/or spleen.

Dosage Forms and Dosing Regimen

[0288] In some embodiments, a composition is administered in a therapeutically effective amount and/or according to a dosing regimen that is correlated with a particular desired outcome (e.g., with treating or reducing risk for sickle cell disease).

[0289] Particular doses or amounts to be administered in accordance with the present invention may vary, for example, depending on the nature and/or extent of the desired outcome, on particulars of route and/or timing of administration, and/or on one or more characteristics (e.g., weight, age, personal history, genetic characteristic, lifestyle parameter, etc., or combinations thereof). Such doses or amounts can be determined by those of ordinary skill. In some embodiments, an appropriate dose or amount is determined in accordance with standard clinical techniques. Alternatively or additionally, in some embodiments, an appropriate dose or amount is determined through use of one or more in vitro or in vivo assays to help identify desirable or optimal dosage ranges or amounts to be administered.

[0290] In various embodiments, a recombinant heme oxygenase protein is administered at a therapeutically effective amount. Generally, a therapeutically effective amount is sufficient to achieve a meaningful benefit to the subject (e.g., treating, modulating, curing, preventing and/or ameliorating the underlying disease or condition). In some particular embodiments, appropriate doses or amounts to be administered may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0291] In some embodiments, a provided composition is provided as a pharmaceutical formulation. In some embodiments, a pharmaceutical formulation is or comprises a unit dose amount for administration in accordance with a dosing regimen correlated with achievement of the reduced incidence or risk of sickle cell disease.

[0292] In some embodiments, a formulation comprising a recombinant heme oxygenase protein or recombinant heme oxygenase-Fc fusion protein described herein administered as a single dose. In some embodiments, a formulation comprising a recombinant heme oxygenase protein or recombinant heme oxygenase-Fc fusion protein described herein is administered at regular intervals. Administration at an “interval,” as used herein, indicates that the therapeutically effective amount is administered periodically (as distinguished from a one-time dose). The interval can be determined by standard clinical techniques. In some embodiments, a formulation comprising a recombinant heme oxygenase protein or recombinant heme oxygenase-Fc fusion protein described herein is administered bimonthly, monthly, twice monthly, triweekly, biweekly, weekly, twice weekly, thrice weekly, daily, twice daily, or every six hours. The administration interval for a single individual need not be a fixed interval, but can be varied over time, depending on the needs of the individual.

[0293] As used herein, the term “bimonthly” means administration once per two months (i.e., once every two months); the term “monthly” means administration once per month; the term “triweekly” means administration once per three weeks (i.e., once every three weeks); the term “biweekly” means administration once per two weeks (i.e., once every two weeks); the term “weekly” means administration once per week; and the term “daily” means administration once per day.

[0294] In some embodiments, a formulation comprising a recombinant heme oxygenase protein or recombinant heme oxygenase-Fc fusion protein described herein is administered at regular intervals indefinitely. In some embodiments, a formulation comprising a recombinant heme oxygenase protein or recombinant heme oxygenase-Fc fusion protein described herein is administered at regular intervals for a defined period.

[0295] As described herein, the term “therapeutically effective amount” is largely determined based on the total amount of the therapeutic agent contained in the pharmaceutical compositions of the present invention. A therapeutically effective amount is commonly administered in a dosing regimen that may comprise multiple unit doses. For any particular composition, a therapeutically effective amount (and/or an appropriate unit dose within an effective dosing regimen) may vary, for example, depending on route of administration or on combination with other pharmaceutical agents.

[0296] In some embodiments, administration of a recombinant heme oxygenase protein or recombinant heme oxygenase-Fc fusion protein reduces the intensity, severity, or frequency, or delays the onset of at least one sickle cell disease sign or symptom. In some embodiments administration of a recombinant heme oxygenase protein or recombinant heme oxygenase-Fc fusion protein reduces the intensity, severity, or frequency, or delays the onset of at least one sickle cell disease sign or symptom selected from the group consisting of vasoocclusion, acute chest syndrome, or organ damage.

[0297] In some embodiments, administration of a recombinant heme oxygenase protein or recombinant heme oxygenase-Fc fusion protein results in improved clinical outcomes as measured by increased real-time peripheral arterial blood oxygen saturation, breath rate, heart rate, pulse distention and reduced lung wet/dry ratio.

TABLE-US-00049 TABLE 2 rHO-1-Fc fusion proteins IgGl Heterodimeric HO-1 FcLALA Mutations on Name Seq Linker Position IgG1 FcLALA Format HO1-Fc- 1-261 no C-terminal Wild- Homodimer v1 linker fusion type Fc bivalent HO1-Fc- 1-261 (GGG C-terminal Wild- Homodimer v2 GS).sub.4 fusion type Fc bivalent linker (SEQ ID NO: 21) HO1-Fc- 1-261 (GGG N- Wild- Homodimer v3 GS).sub.4 terminal type Fc bivalent linker fusion (SEQ ID NO: 21) HO1-Fc- 1-261 no C-terminal Wild- Chain A: monovalent v4 linker fusion type T350V/L351Y/F405A/Y4 07V Chain B: T350V/T366L/K392L/T39 4W HO1-Fc- 1-261 no N- Knob- Chain A: T366W, S354C; monovalent v5 linker terminal Hole Chain B: T366S, L368A, fusion S-S Y407V, Y349C HO1-Fc- 1 -261 no N- DD- Chain A: K392D, K409D; monovalent v6 linker terminal KK Chain B: D399K E356K fusion HO1-Fe- 1-261 no N- EW- Chain A: K360E, K409W, monovalent v7 linker terminal RVT Y349C; fusion S-S Chain B: Q347R, D399V, F405T, S354C HO1-Fc- 1-261 no C-terminal Knob- Chain A: T366W; Chain monovalent v8 linker fusion Hole B: T366S, L368A, Y407V HO1-Fc- 1-261 no C-terminal EW- Chain A: K360E, K409W monovalent v9 linker fusion RVT Chain B: Q347R, D399V, F405T HO1-Fc- 1-226 no C-terminal Chain A: monovalent v10 linker fusion T350V/L351Y/F405A/Y4 07V Chain B: T350V/T3 66L/K3 92L/T3 9 4W HO1-Fc- 1-226 no N- Wild- homodimer Fc v11 linker terminal type fusion HO1-Fc- 1-226 no C-terminal Wild- homodimer Fc v12 linker fusion type HO1-Fc- 1-261 no C-terminal Wild- single chain v13 linker fusion type (G4S).sub.8 (SEQ ID NO: 63) monovalent HO1-Fc- 1-261 no C-terminal K-D Reverse charge mutations: single chain v14 linker fusion Chain A: E357K; Chain B: (G4S).sub.13 (SEQ K370D ID NO: 64) monovalent HO1-Fc- 1-226 no C-terminal Wild- single chain v15 linker fusion type (G4S).sub.8 (SEQ ID NO: 63) monovalent HO1-Fc- 1-226 no C-terminal K-D Reverse charge mutations: single chain v16 linker fusion Chain A: E357K; Chain B: (G4S).sub.13 (SEQ K370D ID NO: 64) monovalent

EXAMPLES

[0298] The following examples describe some of the preferred modes of making and practicing the present invention. However, it should be understood that these examples are for illustrative purposes only and are not meant to limit the scope of the invention.

Example 1. Production of Truncated Recombinant Human Heme-Oxygenase 1 (rHO-1)

[0299] This example illustrates expression of recombinant heme oxygenase (rHO-1). A series of recombinant heme oxygenase (rHO) truncated proteins were engineered with a His-tag at the C-terminal end. Five truncated variants (1-226, 1-233, 1-237, 1-261, 1-265) were assessed for expression in E. coli. All truncated HO-1 constructs showed stable expression and solubility. His-1-261 was selected for further purification and evaluation.

[0300] A 2 L culture of BL21 (DE3) cells expressing rHO-1 was centrifuged and was lysed in PBS buffer containing 5 mM imidazole in the presence of a protease inhibitor. His-tag rHO-1 was affinity purified using Ni-FF XK 16/20 column eluted with 500 mM imidazole PBS elution buffer. rHO-1-His fractions were pooled and desalted.

[0301] rHO-1-His was further purified using a Superdex 75 SEC column. Purified rHO-1 protein was onto Detoxi-Gel Endotoxin Removing Gel column for resulting in a final endotoxin level of 1.10 Eu/mg. Purity was determined to be 95% by SDS-PAGE and a protein yield of 160 mg was achieved in a final concentration of 4.02 mg/ml.

Example 2. Enzymatic Activity of Purified rHO-1

[0302] This example demonstrates enzymatic activity of purified rHO-1. rHO-1 was incubated with hemin, BSA, and catalase enzyme. Rat Kidney Cytosolic Fraction was added to the reaction to provide sufficient biliverdin reductase. The reaction was started by the addition of 1 mM β-NADPH and incubated at 37° C. for 30 minutes. The reaction was stopped by placing on ice.

[0303] Chloroform was vortexed on ice, added to the reaction mixture, mixed 3× in the dark, centrifuged at 1000 rpm for 2 minutes to separate organic phases cleanly. Formed bilirubin was extracted in the chloroform phase and collected from the bottom of the tube into a fresh amber colored tube on ice. Spectrophotometric absorbance was measured between 420-540 nm in a 1 cm path length quartz cuvette.

[0304] Heme oxygenase activity was calculated as bilirubin product formed (nmol)/mg protein/hr using the extinction coefficient of bilirubin as 58 mM/cm. Enzymatic activity of truncated rHO-1 is shown in FIGS. 1A and 1B and Table 3.

TABLE-US-00050 TABLE 3 Enzymatic activity of 1-261-His and His-1-261 Michaelis- Menten (Best 1-261-His fit values) (R&D) 1-261-His His-1-261 Vmax 0.3332 0.3456 0.3523 Km 0.1401 0.1767 0.1874

Example 3. Pharmacokinetic (PK) Profile and In Vivo Efficacy of rHO-1-His in Mice

[0305] This example illustrates that systemic administration of recombinant heme oxygenase (HO-1) in mice showed a trend of increased survival following hemin challenge and amelioration of symptoms of sickle cell disease.

[0306] Transgenic Townes sickle mouse model (SS mice) was used since it recapitulates many of the major pathophysiologic aspects of SCD, in particular vaso-occlusive crises (VOC) and Acute Chest Syndrome (ACS) (See Ghosh et al., 2013. J Clin Invest. 2013:123(11):4809-4820). Free heme triggers Acute Chest Syndrome with severe hypoxemia and death in sickle cell mice. The SS transgenic Sickle cell disease mouse model mimics chronic anemia seen in human disease. Similarly, in SS mice, extracellular heme triggers hemolysis and VOC/ACS.

[0307] Both young (4-6 weeks) and adult (12 weeks or and older) SS mice suffer hemolytic crises when challenged with intravenous hemin (35 micromoles/kg bw). However, the young mice do not develop acute lung injury (ALI) while the adults succumb to respiratory failure. The young mice rapidly cleared excess heme from the circulation suggesting this may be the reason for their resistance to intravenous hemin. Interestingly, the young mice had significantly lower plasma hemopexin levels compared to the adult mice excluding the classical heme scavenging pathway for their resistance.

[0308] SS mice were systemically administered a single dose of 5 mg/kg of 1-261-His or vehicle control intravenously. Blood was collected at 5 minutes, 1 hour, 2 hours, 4 hours, 12 hours and 48 hours after injection of rHO-1-His. and plasma separated for analysis. As shown in FIG. 2, 1-261-His exhibited a one-phase decay with a half-life of 69.4 minutes. A 2 log difference in levels of plasma HO-1 was observed in animals injected with HO-1-His within 48 h. (FIG. 2).

[0309] To evaluate survival in SS Mice following treatment with 1-261-His and heme-induced acute-chest syndrome. Transgenic SS mice with human HbS were systemically injected with 5 mg/kg 1-261-His or vehicle control and monitored for survival following intravenous administration of 35 μmol/kg heme up to 120 minutes after delivery, 75% mice receiving heme oxygenase survived 120 minutes after delivery (FIG. 3).

[0310] Response following in vivo treatment with 1-261-His was monitored by evaluating real time oxygen saturation, breath rate, heart rate, pulse distention and lung wet/dry ratio as surrogate clinical outcomes (FIG. 4A-4E).

[0311] Peripheral arterial oxygen saturation in blood (% SpO2) was measured in real time by pulse oximetry up to 120 minutes following treatment with hemin. Pulse oximetry uses spectrophotometry to determine the proportion of hemoglobin that is saturated with oxygen (i.e., oxygenated hemoglobin; oxyhemoglobin) in peripheral arterial blood. Light, at two separate wavelengths, illuminates oxygenated and deoxygenated hemoglobin in blood. The ratio of light absorbance between oxyhemoglobin and the sum of oxyhemoglobin plus deoxyhemoglobin is calculated and compared with previously calibrated direct measurements of arterial oxygen saturation (SaO2) to establish an estimated measure of peripheral arterial oxygen saturation (SpO2). Pulse oximeter probes consist of two light-emitting diodes and a photodetector. Deoxyhemoglobin absorbs light maximally in the red band of the spectrum (600 to 750 nm), and oxyhemoglobin absorbs maximally in the infrared band (850 to 1000 nm). Thus, the emitters emit light at 660 nm and 940 nm for optimal detection of these two substances. Peripheral arterial oxygen saturation is used as a biomarker for tissue oxygenation. In this exemplary study, increased oxygen saturation was observed in SS mice following a 35 μmol/kg hemin challenge.

[0312] Breath rate per minute was measured in real time was measured by pulse oximetry up to 120 minutes following treatment with hemin. The MouseOx pulse-oximeter (Starr Life Sciences) was used to measure real-time SpO.sub.2 (percentage of functional arterial Hb) and breath rate per minute in awake conditions. Hairs from the collar region (back of the neck) were removed using a depilatory agent 1 day before actual measurement. A disposable sensory collar clip attached to the pulse-oximeter was placed on the hairless area, and measurements were initiated through MouseOx software (version 6.3; provided by the manufacturer) when data displays were without error codes. Recorded values were pooled for each consecutive 5-minute interval, and mean values were used for analysis where continuous screening was presented. Breath rate is derived from respiratory effort and not airflow and will be present even if the animal is experiencing obstructive apnea.

[0313] Heart rate per minute was measured in real time cardiac pulse rate (bpm) up to 120 minutes following treatment with hemin. The MouseOx pulse-oximeter (Starr Life Sciences) was used to carry out measurements. Pulse distention was measured in real time up to 120 minutes following treatment with hemin. Pulse distention is a measurement of the change in distention of the arterial blood vessels residing between the sensors pads due to a cardiac output pulse. It is a direct measurement of changes in local blood volume that accompany each cardiac pulse. For a given vascular compliance, pulse distention can also provide a surrogate for pulse pressure.

[0314] Pulse distention was measured in real time up to 120 minutes following treatment with hemin. Pulse distention is a measure of change in the effect path length of light that passes through the arterial or pulsating blood and has true linear distance unites of μm. Pulse distention provides a measure of arterial blood available to make oximetry measurement for parameters such as blood oxygen saturation, heart rate and breath rate.

[0315] Lung wet/dry ratio was measured up to 120 minutes following treatment with hemin. Higher lung wet/dry weight ratio is indicative of lung edema, and is a biomarker of Acute Chest Syndrome. The whole lungs were harvested from mice, either immediately after death or 2 hours after hemin injection, and weighed using an isometric transducer. Lungs were then dried in an oven at 80° C. containing desiccant crystals for 24 hours, dry weight was determined, and lung wet/dry weight ratios were calculated.

[0316] Mice treated with 1-261-His showed HO-1 level with a half-life of 69.4 min. Mice treated with 1-261-His showed increased survival, increased oxygen saturation, breath rate, heart rate and decreased lung wet/dry ratio in SS mice following a 35 μmol/kg hemin challenge.

Example 4. In Vitro Activity and PK Analysis of HO-1-Fc Fusion Proteins

[0317] This example illustrates the development of HO-1 fusion proteins designed to increase the half-life of recombinant HO-1.

[0318] Four Fc fusion constructs were engineered using truncated rHO-1 protein fused with an Fc domain, 1-261-Fc V1 (SEQ ID NO: 3) was produced using HEK293 cells. V2 (SEQ ID NO: 4), V3 (SEQ ID NO: 5) and V4 (SEQ ID NO: 6) were produced using ExpiCHO cells (FIG. 5A-5E).

[0319] Expression plasmids encoding the 1-261-Fc fusion constructs (V1, V2 and V3) or two plasmids encoding chains A and B respectively (V4) were transfected with PEI. Conditioned medium was harvested 9 days post transfection by centrifugation and filtration, 1-261-Fc proteins were purified from conditioned medium by binding and elution from MabSelect SuRe. Eluates from MabSeclect SuRe were further purified using a Superdex 200 column and 1-261-Fc containing fractions were pooled. Purified protein was analyzed by SDS-PAGE and SEC-HPLC.

[0320] This example illustrates that systemic administration of 1-261-Fc fusion protein in mice showed extended half-life.

[0321] Enzymatic activity of V1, V2, V3 and V4 Fc fusion proteins was measured in vitro. rHO-1 (80 μg/ml) was incubated with hemin (60 μM), BSA (4 mg/mL), Cytochrome P450 Reductase (80 μg/mL), Biliverdin Reductase A (80 μg/mL) and catalase (1000 U/mL). Read absorbance at 468 nm (bottom read) in kinetic mode for 5 minutes at 37° C. after the reaction was started by the addition of 1 mM β-NADPH. 1-261-Fc fusion proteins V1, V2, V3 and V4 had similar activity profiles as 1-261-His protein. V2 and V3 aggregates showed lower activity than the 1-261-His control (FIG. 6).

[0322] To evaluate the pharmacokinetic profile of the HO-1 Fc fusion proteins, WT CD1 mice (4 mice per time points) were administered intravenous injections of V2, V3, V4 and 1-261-His. Plasma rHO-1 levels were measured by ELISA assay as described in Example 3. As shown in Table 4, V2, V3 and V4 demonstrated showed extended half life and were detectable at 168 hours and 1-261-His had a half-life of approximately 40 min (0.67 h) (FIG. 7).

TABLE-US-00051 TABLE 4 In vivo PK parameters of rHO-I proteins Terminal C.sub.0 AUC.sub.0-168 hr. Clearance Construct half-life (hr) (μg/mL) (hr* ug/mL) (mL/r/kg) 1-261-Fc V2 85 84 3321 1.15 1-261-Fc V3 92 129 5053 0.730 1-261-Fc V4 101 128 5568 0.641 1-261-His 0.67 173 15.9 14

Example 5. In Vivo Efficacy of Fc Fusion Heme Oxygenase Truncated Proteins

[0323] This example illustrates the in vivo efficacy of recombinant heme oxygenase Fc fusion proteins in increasing survival in mice that received the treatment. Young SS mice have 2-fold higher plasma HO-1 than adult mice. Fractionization experiments showed that HO-1 co-localizes with the enzymatic and co-factor machinery required for heme degradation in the plasma affirming that heme can be degraded in the plasma. To determine whether young SS mice use HO-1 to rapidly degrade circulating heme, 3 week old animals were treated with an HO-1 inhibitor (tin protoporphyrin (SnPP)) or vehicle. Seven days later, the animals were challenged with hemin to induce ALI/ACS. A majority ( 10/12: 83%) of the vehicle-treated mice survived, while the SnPP-treated mice ( 10/14; 71%) developed lethal ALI/ACS.

[0324] Young SS mice were treated for three months through adulthood with Nrf2 activator to stimulate HO-1 expression, and then treated with vehicle or SnPP prior to ALI/ACS induction. The SnPP-treated group developed lethal ALI/ACS while the vehicle treated SS mice with elevated HO-1 in adulthood survived. Finally, adult SS mice were concomitantly induced to develop ALI/ACS and infused with novel HO-1 recombinants or vehicle. The HO-1 recombinants attenuated lung injury and improved survival with one variant V4 affording 100% protection among a cohort of adult SS mice that would normally succumb to respiratory failure at a lethality rate of 70%.

[0325] SS mice were administered V3 (9.2 mg/kg) and V4 (13.9 mg/kg) at equivalent molar amounts to 1-261-His (5 mg/kg). Survival in mice was measured up to 120 minutes after intravenous hemin challenge to induce ACS. 100% of mice that received 1-261-Fc V3 or 1-261-Fc V4 survived (FIG. 8A) while only 20% mice that received vehicle control survived.

[0326] Blood oxygen saturation, lung wet/dry weight ratio, breath rate per minute, heart rate per minute and pulse distention were measured following intravenous challenge with hemin. Peripheral arterial blood oxygen saturation was significantly increased following administration of 1-261-Fc V3 or 1-261-Fc V4 treatment relative to a vehicle control (FIG. 8B). Mice treated with 1-261-Fc V3 (outliers included 2 visibly sick mice with low Sp02) and 1-261-Fc V4 showed reduced lung wet/dry ratio (FIG. 8C).

[0327] Mice treated with 1-261-Fc V3 or 1-261-Fc V4 showed increased heart rate of about 700 bpm, relative to vehicle treated controls that measured 600 bpm (FIG. 9A). Mice treated with 1-261-Fc V3 or 1-261-Fc V4 showed sustained breath rate of about 125 bpm relative to vehicle treated controls (FIG. 9B). Mice treated with 1-261-Fc V4 showed increased and sustained pulse distention of about 600 μm relative to vehicle-treated control mice. Treatment with 1-261-Fc V3 showed sustained pulse distention as compared to vehicle-treated control mice but only at about 300 μm (FIG. 9C).

[0328] Mice treated with 1-261-Fc V3 or V4 showed improved survival after hemin challenge relative to a vehicle control. In addition, there was increased blood saturation oxygen and decreased lung wet/dry weight ratio in mice treated with recombinant heme oxygenase. Treatment with V3 or V4 also improved heart rate and breath rate in mice. V4 treatment also improved pulse distention.

Example 6. rHO-1 Enzyme Kinetics

[0329] This example illustrates use of a sensitive assay for measuring rHO-1 enzyme kinetics. rHO-1 catalytic activity can be measured in an enzyme linked assay measuring bilirubin absorbance. However, enzyme linked assay measuring bilirubin absorbance assays requires a large amount of product and reagent in microgram amounts. Further, dose-dependent linearity and batch to batch reproducibility remain challenging. A fluorescent assay using UnaG, which is a 16 kDa fluorescent protein from Japanese eel muscle that binds Bilirubin with 1:1 stoichiometry to create a complex that excites at 480 nm and emits at ˜520 nm was used to determine enzyme kinetics of the rHO-1-Fc constructs. This provides a sensitive reporter for detection of product to assess rHO-1 enzyme kinetics (FIG. 11).

[0330] His-tagged UnaG was expressed in E. coli from an expression vector and purified by affinity purification using a nickel column and dialysis to about >90% pure. Assay conditions included 100 nmol HO-1-Fc construct, 10 μM Hemin+BSA, 250 μM NADPH, 0.5 μM Biliverdin reductase, 2 μM UnaG in a 10 μL assay volume. The UnaG assay demonstrated dose dependent activity for HO-1 (FIG. 12A) and does not have background fluorescence (FIG. 12B).

[0331] Heme oxygenase (HO) degrades heme in concert with NADPH cytochrome P450 reductase (CPR) which donates electrons to the reaction. P450 oxidoreductase (POR) transports electrons from NADPH to cytochrome P450. A molar comparison of rHO-1-Fc constructs with or without P450 cytochrome (POR)/catalase was performed as shown in FIG. 13A (with catalase and POR) and 13B (without catalase and POR).

[0332] As shown in Table 5, the average rate of HO-1 enzyme activity measured in Fluorescent Units/minute without catalase and without POR were between 60-80 min. The average rate of Fluorescent Units/minute with catalase and with POR are calculated between 0-20 min. and values shown in Table 5. These results suggest that the assay is highly reproducible, used much less reagent, and can be conducted in a high throughput manner.

TABLE-US-00052 TABLE 5 Average rate of HO-1 Enzyme Activity (Fluorescent Units/minute) Without catalase and POR With catalase and POR Sample Av Rate FU/min Av Rate FU/min SD 1-261-His — 342.32 45.27 V1 55.23 880.22 74.11 V2 86.15 1815.81 232.02 V3 44.13 477.60 45.43 V4 22.53 382.31 21.72 V4 (10 L prep) 25.96 306.14 39.11 V5 17.90 217.80 26.23 V6 16.12 221.54 39.68 V7 19.39 241.70 51.72 V8 4.73 35.15 5.07 V9 28.60 386.86 63.07 V10 22.80 283.54 24.00 V11 17.93 150.15 2.68 V12 40.83 469.79 40.68 V15 14.45 180.76 5.31

Example 7. In Vivo Efficacy of rHO-1(1-261)-Fc V4 Fusion Heme Oxygenase Truncated Protein

[0333] This example illustrates in vivo efficacy of different doses of recombinant heme oxygenase Fc fusion proteins in increasing survival in mice that received the treatment.

[0334] To determine the efficacy of rHO-1-Fc V4, SS mice were administered a single intravenous dose of rHO-1-Fc v4 of either 1.4 mg/kg, 4.7 mg/kg or 13.9 mg/kg (equimolar to 1/10, ⅓, or a total 5 mg/kg dose of rHO-1 [1-261]-His respectively) or carrier vehicle. Survival in mice was measured up to 120 minutes after intravenous hemin challenge to induce ACS. In this study, about 80% of mice that received 13.9 mg/kg (equimolar to 5 mg/kg dose) 1-261-Fc V4 survived and about 40% of mice that received 4.7 mg/kg (⅓) survived while only about 30% mice that received vehicle control survived (FIG. 10A).

[0335] SS mice were given a single intravenous dose of rHO-1-Fc V4 of 13.9 mg/kg (equimolar to a 5 mg/kg dose of rHO-1 [1-261]-His) or carrier vehicle. Survival in mice was measured up to 120 minutes after intravenous hemin challenge to induce ACS. 90% of mice that received rHO-1-Fc V4 survived (FIG. 10B) while only 25% of mice that received vehicle control survived.

[0336] Blood oxygen saturation, lung wet/dry weight ratio, breath rate per minute, heart rate per minute and pulse distention were measured following intravenous challenge with hemin. Peripheral arterial blood oxygen saturation was significantly increased following administration of 1-261-Fc V4 treatment relative to a vehicle control for mice administered with 13.9 mg/kg or 4.7 mg/kg V4 (FIG. 10C). Vehicle data, and data for mice administered 4.7 mg/kg or 1.4 mg/kg V4 were from SS mice that expired within 2 h following of hemin infusion. Mice treated with 13.9 mg/kg or 4.7 mg/kg rHO-1-Fc V4 showed reduced lung wet/dry ratio relative to mice treated with a vehicle control (FIG. 10D).

Example 8. rHO-1(1-261)-Fc Chimeras with Reduced Aggregation

[0337] rHO (1-261)-Fc chimeric constructs were developed with improved properties compared to recombinant HO-1 alone (FIG. 5A-5Q). Constructs were designed to improve batch to batch reproducibility in CHO cell lines without post-translational modifications. Additionally, the rHO-1 Fc fusion variants demonstrate reduced aggregation of full length (1-261) rHO-1-Fc during affinity purification and SEC in PBS.

[0338] Constructs v1-v16 were transiently transfected and expressed in 100 mL EBNA1 cells. In an exemplary expression protocol, 1:1 ratios of each Fc chain, sheared salmon sperm DNA (filler DNA) and XBP1S (co-expressed to improve folding and secretion) were loaded onto a Maxcyte CL 1.1 cassette and electroporated. 48 hours post-transfection, 0.125% N,N Dimethylacetamide was added and cell feed with 3% SAFC Advanced Feed 1 was provided daily until day 8. Constructs are purified using a citrate gradient followed by a polishing step on SEC in PBS.

[0339] Constructs were screened and selected if >95% purity was achieved after two rounds of purification and were resistant to thermal stress (3× Freeze Thaw analysis). Enzymatic activity of heme breakdown was tested in in vitro assays to identify constructs that achieved similar robustness and activity relative to cell free (cf) HO-1. As shown in Table 6, HO-1 activity of rHO-1-Fc constructs was assessed relative to the V4 construct. V12, V10, and V7 demonstrated robust enzymatic activity and were purified with high yield.

TABLE-US-00053 TABLE 6 Enzymatic fold difference of rHO-1 constructs relative to V4. rHO-1-Fc Enzymatic Fold diff. to V4 Enzymatic Fold diff, to V4 10 L Name 10 L prep, mol equivalent prep. HO-1 valency equivalent V1 0.47 0.94 V2 0.3 0.6 V3 0.59 1.18 V4 1.15 1.15 V5 1.45 1.45 V6 1.61 1.61 V7 (3) 1.34 1.34 V8 5.49 5.49 V9 0.91 0.91 V10 (2) 1.14 1.14 V11 1.45 2.9 V12 (1) 0.64 1.27 V15 1.8 1.8

Example 9. Pharmacokinetic Profile of rHO-1 V7, V10 and V12 Fusion Proteins

[0340] To evaluate the pharmacokinetic profile of the rHO-1-Fc fusion proteins, male C57BL/6 (Jax) mice (4 mice per time points) were administered intravenous injections of 5 mg/kg V7, V10, V12 or V4 (control). Serum rHO-1 was collected at 5 m, 6 h, 24 h, 72 h, 168 h, 240 h and 336 h. Plasma rHO-1 will be measured by ELISA assay.

Example 10. rHO-1-Fc V4 Fusion Protein In Vivo

[0341] This example illustrates administration of rHO-1-Fc V4 fusion protein in an HbSS and an HbAA mouse model. Male and female HbSS and HbAA mice are intravenously injected with rHO-1-Fc V4 fusion protein or PBS as a control weekly. HbSS mice are intravenously administered with a weekly dose of 13.9 mg/kg of HO-1, 5 mL/kg of HO-1 V4. Response following in vivo treatment with rHO-1 V4 are monitored by evaluating real time oxygen saturation, breath rate, heart rate, pulse distention and lung wet/dry ratio as surrogate clinical outcomes, as described in Example 3.

[0342] Blood for hematological assessment and organs for histopathology, immunohistochemistry and quantitative plasma assays are collected at 10 weeks, 12 weeks, 16 weeks and 20 weeks as shown in Table 7. Hematology endpoint assessments will measure hemoglobin, red blood cells mean corpuscular volume (MCV), white blood cells and differential, reticulocytes, % sickle erythrocytes, w/ microscopic pictures, spo.sub.2, heart rate, plasma free heme, plasma free hemoglobin, plasma hemopexin, plasma haptoglobin, and d-dimer quantification. Histopathology studies will assess liver, lung (no lavage), spleen including weight (% body weight), kidney, brain, and heart. Immunohistochemistry analysis will be performed to evaluate H&E, iron, p-selectin, ICAM, V-CAM, lymphocytes, oxidative stress markers and kidney depot C3/C5. Quantitative plasma assays will be used to measure ferritin, iron, transferrin, bilirubin, AST, ALT, LDH, NO, and pro-inflammatory cytokines using multiplex cytokine release. Pharmacokinetic studies of rHO-1 V4 fusion protein administration are performed in HbAA mice from groups 11 and 12.

TABLE-US-00054 TABLE 7 Timeline for blood and organ collection Time-point for blood Group Genotype Injection and organ collection 0 HbSS PBS 10 weeks 1 HbSS PBS 12 weeks 2 HbSS 13.9 mg/kg HO-1 12 weeks V4: 5 mL/kg 3 HbSS PBS 16 weeks HbSS 13.9 mg/kg HO-1 16 weeks V4: 5 mL/kg 5 HbSS PBS 20 weeks HbSS 13.9 mg/kg HO-1 20 weeks V4: 5 mL/kg 7 HbAA PBS 10 weeks 8 HbAA PBS 12 weeks 9 HbAA PBS 16 weeks 10 HbAA PBS 20 weeks 11 HbAA 13.9 mg/kg HO-1 2 days, week 11, 13, V4: 5 mL/kg 15, 17, 19 12 HbAA 13.9 mg/kg HO-1 2 h, 4 days, week V4: 5 mL/kg 12, 14, 16, 18, 20
Toxicity Profile of rHO-1-Fc V4 Fusion Protein

[0343] To measure toxicity of rHO-1-Fc V4 fusion protein, 14-16 wk old HbSS and HbAA mice (mature SCD phenotype) are administered a single escalating dose rHO-1-Fc V4 selected from the following doses: 0, 15, 50 or 150 mg/kg. 6 mice per sex will be administered rHO-1 at each dose tested in the study. After 2 h intravenous exposure to rHO-1-Fc bilirubin toxicity endpoints are measured followed by serum and tissue iron levels at 7 days post injection.

TABLE-US-00055 TABLE 8 Conditions for determining in vivo toxicity profile of rHO-1-Fc V4. rHO-1-Fc V4 Volume Concentration Group Genotype (mg/kg) (mL/kg) (mg/mL) 1 HbSS 0 5 0 2 HbSS 15 5 3 3 HbSS 50 5 10 4 HbSS 150 5 30 5 HbAA 0 5 0 6 HbAA 150 5 30

Example 11. HO-1 Levels Associate with Risk of ACS in SCD Patients

[0344] Previous genetic association studies have linked high heme oxygenase-1 (HO-1) expression with low ACS risk in children. SCD patients were evaluated for HO-1 levels. The present study shows for the first time that the concentration of plasma HO-1 in SCD children 1-9 yrs is 2-fold higher than adults 20 yrs and older (23.6±1.1, n=191 versus mean 10.7±0.6, n=67).

INCORPORATION BY REFERENCE

[0345] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. 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. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

EQUIVALENTS AND SCOPE

[0346] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims.