Oxygen-binding chimeric proteins

Abstract

The present invention describes chimeric proteins having albumin or truncated albumin covalently linked to one or more oxygen-binding proteins or protein fragments or variants, their preparation and use

Description

OXYGEN-BINDING CHIMERIC PROTEINSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 726,601 filed on December 1, 2024 and U.S. Provisional Application No. 63,793,941 filed on April 24, 2025, both of which are incorporated by reference in their entirety.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY:

[0002] The content of the electronically submitted sequence listing (Name: 9170_0101_Sequence_Listing.xml; Size: 107,653 bytes; and Date of Creation: November 24, 2025) filed with the application is incorporated herein by reference in its entirety.FIELD OF THE INVENTION

[0003] The present invention describes chimeric proteins having albumin or truncated albumin covalently linked to one or more oxygen-binding proteins or their variants, their preparation and use.BACKGROUND OF THE INVENTION

[0004] Blood transfusion is a standard procedure used to treat patients with acute anemia, such as major blood loss due to surgery, injury, traumatic hemorrhagic shock. It is also a last resort for patients suffering from chronic blood disorders, including sickle cell disease, thalassemia, aplastic anemia, autoimmune hemolytic anemia (AIHA), etc. Every year, there are millions of patients who suffer or die from severe anemia or blood loss, where blood transfusion is incompatible or unavailable (Chambers 2019). Serious side effects from blood transfusion using incompatible blood often range from severe immune reaction to allogeneic blood to death often occur. Furthermore, shortage of donated blood, inaccessible to hospital or emergency care at times of accident, natural disasters or war are recurrent issues. As a result, there is high unmet medical need for a stable and safe replacement for donor blood and in prehospital or emergency settings.

[0005] For almost a century, researchers have been looking for a safe and ready to use alternative to human blood transfusions. For anemia, major research efforts have been centered around oxygen carrying molecules: hemoglobin-based oxygen carriers (HBOC) and Perfluorocarbons (PFC) (Gupta 2019, lahr 2021). In spite of these efforts, as of today, a safe, viable blood substitute product remains elusive, due to formulation difficulties, unexpected toxicities, and severe side-effects that encountered by replacement blood products in development (Alayash 2019, Jahr Aug 2022). Forexample, the adverse events have been observed in clinical trials. These effects could impact cardiac, gastrointestinal, hepatic, pancreatic, central nervous, and renal systems. Clinical symptoms often include kidney, spleen, liver toxicities, hypertension, myocardial infarction, and even death.

[0006] It has been found that cell-free hemoglobin from HBOC dissociated into monomeric and dimeric forms have very short half-life in circulation, accumulated in kidney, spleen, and liver and causing organ damages (Buehler, et al. 2010). Besides short half-life, administration of cell-free HBOC leads to imbalance of osmotic pressure and volume in the blood (Gupta 2019). Similar effects are also seen with the next generation encapsulated HBOCs.

[0007] Recent studies have shown that the multi-system toxicities may be also linked to HBOC- mediated extravasate nitric oxide scavenging, hyperoxygenation, and heme-mediated toxicity (Alayash 2019). Hemoglobin binds not only to oxygen but also several other molecules, such as nitric oxide (NO). Leaked free form of Hb could sweep up NO within the subendothelial layer of blood vessels, resulting in low NO level, vasoconstriction, hypertension, and damage to various organs.

[0008] When hemoglobin undergoes autooxidation, it can release its heme group, an iron- containing molecule. This process generates reactive oxygen species (ROS), leading to potential oxidative stress. Oxidative stress can damage cell membranes, proteins, and DNA. This autooxidation may be particularly significant in concentrated cell-free hemoglobin-based oxygen carriers (HBOCs) which lack the natural reductive enzymes found in red blood cells.

[0009] Human serum albumin (HSA, molecular weight 65kDa) is the most abundant protein in plasma blood and has a long half-life (up to 21 days) circulation. Due to its negative charge at physiological pH, albumin generally cannot cross a cell membrane under normal conditions. It serves various and vital functions, such as regulates oncotic pressure and volume of blood, binds and transports molecules (fatty acid, steroid, heme, metal ions, drugs, etc.). It also serves scavenger and antioxidant roles in blood. Albumin is recognized for its antioxidant properties, which enable it to neutralize various ROS and reactive nitrogen species (RNS). Additionally, albumin helps decrease the likelihood of reactive species production by binding to free copper ions (Cu (II)), a primary contributor to free radical formation (Raoufinia 2016).

[0010] Several human serum albumin (blood derived or recombinant) products have been approved by the FDA (Albuminex, Albumin-Human, Kedbumin) and regulatory authorities fromother countries for use to treat patients with hypovolemia, ascites, hypoalbuminemia including from burns, acute nephrosis, acute respiratory distress syndrome, cardiopulmonary bypass (FDA labels).

[0011] The present invention describes a new approach in developing a viable blood replacement therapy, utilizing the complementing properties of albumin to address or alleviate the adverse effects previously observed in developing HBOC. As a result, the chimeric proteins of the present invention have the potential to provide a simple, safe, off-the-shelf solution to patients in urgent need of blood transfusion, especially in emergency surgery, prehospital or remote settings where donated blood is not available. They are optimized to transport oxygen in circulation and deliver oxygen to vital organs and tissues, including heart and brain, without major side effects. They may also serve as blood volume expanders to restore oncotic-osmotic pressure. These synergistic effects make the chimeric proteins of the present invention a true option as a safe substitute for natural blood.

[0012] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.SUMMARY OF THE INVENTION

[0013] The present invention describes engineered, isolated, non-naturally occurring, or recombinant chimeric proteins comprising albumin or truncated albumin or albumin variant covalently linked to one or more oxygen binding protein (s), their truncated version, fragments or variants. The present invention further describes compositions comprising such chimeric proteins, applications for the compositions, methods of use, and methods of preparation.

[0014] One embodiment of the invention is an engineered or isolated chimeric protein comprising albumin, preferably human serum albumin (“HSA” or “Hsa”), covalently linked to an oxygen binding protein, preferably human hemoglobin, myoglobin, neuroglobin or cytoglobin. Albumin can be linked to an oxygen binding protein either at its C-terminal or N-terminal with or without an optional linker. Examples of such chimeric proteins can be found in Figure 1.

[0015] Another embodiment of the invention is an engineered or isolated chimeric protein comprising one albumin, preferably HSA, covalently linked to two oxygen-binding proteins, preferably human hemoglobin, myoglobin, neuroglobin or cytoglobin, in tandem at its C-terminal or N-terminal via one or two optional linkers. Examples of such chimeric proteins can be foundin Figure 2. It is also possible for albumin to have oxygen-binding proteins at both C-terminal and N-terminaL

[0016] Another embodiment of the invention is an engineered or isolated chimeric protein comprising albumin, preferably HSA, covalently linked to at least one oxygen-binding protein at C-terminal via an optional linker and covalently linked to at least one oxygen-binding protein at N-terminal via an optional linker, wherein the oxygen binding protein, is preferably human hemoglobin, myoglobin, neuroglobin or cytoglobin. Examples of such chimeric proteins can be found in Figure 2. It should be noted that, when two or more oxygen-binding proteins are included in a chimeric protein, oxygen-binding proteins may be the same or different from each other, such as one oxygen-binding protein may be hemoglobin and another may be myoglobin.

[0017] One preferred embodiment is an engineered chimeric protein comprising one human serum albumin covalently linked to two or more oxygen-binding proteins, including truncated oxygenbinding proteins or their fragments in tandem at its C-terminal with or without an optional linker. Some of the preferred examples are shown in Figure 3 and Figure 18.

[0018] It should be noted that human hemoglobin subunits as used herein throughout the present application can be one or more human hemoglobin subunits, including subunit alpha, beta, gamma, zeta, epsilon or their variants. Examples of human hemoglobin subunit sequence are shown in Table 3.

[0019] Yet another embodiment of the invention is an engineered chimeric protein comprising one albumin, preferably HSA, covalently linked to three oxygen-binding proteins, preferably human hemoglobin, myoglobin, neuroglobin or cytoglobin, or their truncated versions or fragments or variants in tandem at its C-terminal or N-terminal. Examples of such chimeric proteins can be found in Figure 4 and Figure 19. It is also possible for albumin to have oxygen-binding proteins at both C-terminal and N-terminal.

[0020] It should be noted that, when three oxygen-binding proteins, including their truncated versions or fragments or variants, are included in a chimeric protein, oxygen-binding proteins may be the same or different from each other, such as one oxygen-binding protein may be hemoglobin subunit and another may be myoglobin.

[0021] Another preferred embodiment of chimeric protein of the present invention comprising one albumin, preferably HSA, covalently linked to three oxygen-binding proteins, preferably human hemoglobin, myoglobin, neuroglobin or cytoglobin. It is possible for albumin to have oxygen-binding proteins at C-terminal or N-terminal or both, having the following structures represented in Figure 20.

[0022] Another embodiment of the invention is an engineered chimeric protein comprising a truncated version of albumin, preferably HSA, covalently linked to an oxygen binding protein, preferably human hemoglobin, myoglobin, neuroglobin or cytoglobin. Some of the preferred examples of such engineered chimeric protein are shown in Figure 5, which shows the deletion of certain albumin’s functional domain. Sometimes, it is also possible to delete or truncated multiple albumin’s functional domains. Some examples are shown in Figure 6. It should be noted that the chimeric proteins of the present invention also include any partial deletion(s) of a functional domain of albumin as long as the oxygen-binding and carrying properties are not substantially altered or affected.

[0023] Another embodiment is a chimeric protein having a truncated albumin covalently linked to two or more oxygen-binding proteins or protein fragments in tandem at its C-terminal or N- terminal, or to have an oxygen binding protein to be linked at both C-terminal and N-terminal of a truncated albumin.

[0024] More specifically, some embodiments of the present invention are recombinant or engineered nucleic acid sequences comprise SEQ ID NOs 1-6 as shown in Table 1, or a nucleic acid sequence having at least 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% sequence identity to one of these sequences.

[0025] TABLE 1

[0026] Some further embodiments of the present invention are isolated, recombinant or engineered chimeric proteins comprising amino acid sequences of SEQ ID Nos 7-29 as shown in Table 2, ora protein or polypeptide comprising a sequence having at least 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% sequence identity to one of these sequences.

[0027] TABLE 2

[0028] It should be noted that some of the examples in Table 2 contain one or more optional glycine linker with sequence of (GGGGS)n (SEQ ID NO: 63) where n can be 1 to 7, some of which is underlined. As explained herein, the use of linker may be optional. It is possible that other peptide linkers may be used to replace such linkers.

[0029] It should be further noted that some of the examples may contain a short peptide sequence at its N- or C-terminal. Such sequences may facilitate production, isolation and / or purification of a protein, or may be residues from protein process. Such peptide tags should not affect the overall activities of the chimeric proteins of the protein invention. Thus, the chimeric proteins of the invention may further comprise such peptide tags at its N- or C-terminal. One example is the example of Sequence No. 12, which contains eight histidine residues at the C-terminal. Additional histidine or fewer histidine residues are also possible. Other peptide tags, such as LDGG, may also be used which is the residue from protein process. It should be noted that the use or additional of such amino acid tags is optional.

[0030] It should also be noted that the chimeric proteins of the present invention may be further modified, such as through glycosylation and pegylation. Accordingly, one embodiment of thepresent invention is a pegylated or glycosylated chimeric protein described herein. The chimeric proteins of the present invention may also be modified or linked via non-covalent interactions which are well-known in the art.

[0031] Another aspect of the present invention is an isolated, recombinant or engineered vector or plasmid comprising a nucleic acid sequence encoding a chimeric protein described herein. Such nucleic acid sequences are preferably linked or controlled by a suitable promoter that enables protein expression. One preferably embodiment is a plasmid vector that can be used in eukaryotic cells, preferably in CHO and human cells. One example of such a vector is shown in Figure 7.

[0032] One embodiment of the present invention is an isolated, recombinant or engineered vector or plasmid comprising a nucleic acid sequence selected from SEQ ID NOs: 1-6 as shown in Table 1, or the nucleic acid comprises a sequence having at least 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% sequence identity to one of these sequences.

[0033] One embodiment of the present invention is an isolated, recombinant or engineered vector or plasmid that encodes a chimeric protein or polypeptide sequence of the present invention. One preferred embodiment of the present invention is a recombinant vector or plasmid that encodes a chimeric protein or polypeptide sequence comprising at least one of SEQ ID NO: 7-29 as shown in Table 2 or at least one of SEQ ID No. 54-62 as shown in Table 5 below, or the protein or polypeptide sequence having at least 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% sequence identity to one of these sequences.

[0034] According to another aspect of the present invention, the present invention relates to an isolated, recombinant or engineered cell comprising a vector or an expression vector that encodes at least one a chimeric protein comprising a full length or truncated version of albumin covalently linked to at least one oxygen binding protein, or fragment of such oxygen binding protein. One embodiment of the present invention is an isolated, recombinant or engineered cell comprising a vector, plasmid, or nucleic acid sequence that encodes or is able or encodes a chimeric protein or polypeptide of the present invention. One of the preferred embodiment is an isolated, recombinant or engineered cell comprising a vector or plasmid that encodes or is able or encode a chimeric protein or polypeptide whose sequence is selected from SEQ ID NO: 7-29, 54-62, or the protein or polypeptide sequence having at least 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%,99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% sequence identity to one of these sequences.

[0035] In one embodiment, the cell is a bacteria cell. In one embodiment, the cell is a yeast cell. In one embodiment, the cell is a plant cell. In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a human cell. In one embodiment, the cell is a CHO, Vero and hamster cell.

[0036] According to another aspect of the present invention, the present invention relates to a method of treating or preventing a disease or condition in a subject in need thereof, comprising administering an effective amount of the chimeric protein described in the present invention. One embodiment is a method of treating or preventing a disease or condition in a subject in need thereof, comprising administering a therapeutically effective dose of the chimeric protein(s) described in the present invention.

[0037] In one embodiment, the method of the present invention may be used to treat or prevent one or more disease or condition comprising ischemic stroke, blood transfusion, hemorrhage shock, hemoglobinopathy, hemolytic disorders, sepsis, trauma, anemia, dialysis, traumatic brain injury, myocardial infarction, pulmonary hypertension, cystic fibrosis, hypovolemia, ascites, hypoalbuminemia including from bums, acute nephrosis, acute respiratory distress syndrome, cardiopulmonary bypass, preservation of organ transplant.

[0038] One embodiment of the presentation is a method of blood transfusion, comprising administering to a subject in need of blood transfusion an effective amount of the chimeric protein described herein.

[0039] Another embodiment of the present invention is a method of treating or preventing a disease or condition in a subject in need thereof comprising i) administering a therapeutically effective amount of a chimeric protein described herein, and ii) administering either simultaneously or sequentially an effective amount of another biologically active component,Such other biological active components could comprise platelet, white blood cell, antibiotics, and other components or components which are compatible with the chimeric proteins of the present invention. It is also possible that such other component(s) may further facilitate or reinforce the oxygen binding or carrying ability of the chimeric proteins of the present invention.

[0040] As described, human serum albumin has been approved for treating, preventing or alleviating a number of diseases or conditions previously. Another embodiment of the present invention is a method of treating, alleviating or preventing such disease or condition which albumin has been approved or shown to be useful or effective, with reduced side effects, in a subject in need thereof. One preferred embodiment is a method of treating or preventing hypovolemia, ascites, hypoalbuminemia including from bums, acute nephrosis, acute respiratory distress syndrome or cardiopulmonary bypass while reducing side effects, such as anemia.

[0041] Blood circulation has been linked to aging and aging-related diseases and conditions. It has been shown that fresh blood infusion from young subjects could treat, prevent, or alleviate age-related, undesirable diseases or conditions. Thus, another embodiment of this present invention is a composition for treating, preventing or alleviating age-related symptoms or conditions, comprising an effective amount of at least one chimeric protein described here. Such age-related symptoms and conditions include, without limitation, age-related anemia, age-related blindness, dementia, chronic pain, amnesia and bone-marrow disorder.

[0042] Yet another embodiment is a composition that delays the onset of aging or age-related symptoms or conditions. Accordingly, another embodiment of the present invention is a method of treating, preventing, alleviating or delaying aging and / or age-related symptoms by administering a composition having an effective amount of a chimeric protein described herein.

[0043] Yet another embodiment is a method of delaying the onset of aging or age-related symptoms and conditions comprising a composition of the present invention, having an effective amount of at least one chimeric protein described herein. Yet another embodiment is a method of promoting and facilitating longevity comprising administering an effective amount of at least one chimeric protein described herein.

[0044] A further embodiment of the present invention is a composition comprising an effective amount of at least one chimeric protein of the present invention for preventing, alleviating or delaying aging and / or age-related symptoms, or for delaying the onset of aging or age-related symptoms and conditions, or for promoting and facilitating longevity.

[0045] There are different ways to administer the composition of the present invention in a subject. In one embodiment, the administration is intravenous. Intravenous administration can be onetime, multiple times, periodic or continuous. In one embodiment, the administration isintramuscular. In one embodiment, the administration is intranasal. In one embodiment, the administration is intradermal. In one embodiment, the administration is by an oral bait drop.

[0046] In one embodiment, the subject is humans. In one embodiment, the subject is a mammal. In one embodiment, the subject is a rodent or primate. In one embodiment, the primate is a bonobo, chimpanzee, gibbon, gorilla, human, monkey, or orangutan.

[0047] Another aspect of the present invention is a nucleic acid sequence that comprises the coding sequences of a chimeric protein of the present invention, wherein the nucleic acid sequence, when transfected into a cell or a suitable host, is able to express or produce the chimeric protein. One preferred example of such nucleic acid sequence is mRNA. Such mRNA sequence may further comprise other nucleic acid moiety or moieties, such as LNP, that protect or stabilize the nucleic acid sequence. Thus, another embodiment of the present invention is mRNA sequence comprising the coding sequence of a chimeric protein of the present invention.

[0048] Another aspect of the present invention is a pharmaceutical composition comprising an effective amount of at least one chimeric protein of the present invention, and a pharmaceutically acceptable carrier or diluent.

[0049] One embodiment of the present invention is a pharmaceutical composition comprising an effective amount of at least one chimeric protein of SEQ ID NO: 7-29 and 54-62, and a pharmaceutically acceptable carrier or diluent.

[0050] In one embodiment, the pharmaceutical composition may further comprise an effective amount of another biologically active component. Examples of such biologically active components include, without limitation, white blood cells, platelets, antibody, antibiotic and other small molecules or biologicals.

[0051] In one embodiment, the composition is an engineered or man-made blood substitute composition.

[0052] Due to their chimeric nature which possesses certain properties of albumin and oxygenbinding protein, the chimeric proteins of the present invention should have extended half-life which enables broad range of applications with less endothelial leakage, oxidative effects, potential organ damage. Thus, another embodiment of the presenting invention is a pharmaceutical composition having reduced side effects and / or prolonged shelf-life. Accordingly, another embodiment of the present invention is an optimized pharmaceutical compositioncomprising an effective amount of at least one chimeric protein of the present invention, which are suitable as blood volume expander to restore oncotic-osmotic pressure.

[0053] Accordingly, it is an object of the invention not to encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U.S.C. §112(a)) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product. It may be advantageous in the practice of the invention to be in compliance with Art. 53(c) EPC and Rule 28(b) and (c) EPC. All rights to explicitly disclaim any embodiments that are the subject of any granted patent(s) of applicant in the lineage of this application or in any other lineage or in any prior filed application of any third party is explicitly reserved. Nothing herein is to be construed as a promise.

[0054] It is noted that in this disclosure and particularly in the claims and / or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of’ and “consists essentially of’ have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention. These and other embodiments are disclosed or are obvious from and encompassed by the following detailed description.BRIEF DESCRIPTION OF THE DRAWINGS AND FIGURES

[0055] In the event that the patent or application file contains at least one drawing executed in color, copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings.

[0056] FIG. 1 describes examples of chimeric protein of the present invention wherein the oxy genbinding protein is covalently linked to albumin via an optional linker. The oxygen-binding protein can be either on the N-terminal or C-terminal.

[0057] FIG. 2 describes examples of chimeric protein of the present invention wherein two oxygen-binding proteins are covalently linked to albumin via two optional linkers. The oxygenbinding proteins can be either on the N-terminal or C-terminal, or both.

[0058] FIG. 3 describes preferred examples of chimeric protein of the present invention wherein two oxygen-binding proteins (human cytoglobin, or myoglobin, or neuroglobin) are covalently linked to human serum albumin via optional linkers at its C-terminal.

[0059] FIG. 4 describes examples of chimeric protein of the present invention wherein three oxygen-binding proteins are covalently linked to albumin via three optional linkers. The oxygen- binding proteins can be either on the N-terminal or C-terminal, or both.

[0060] FIG. 5 describes examples of chimeric protein of the present invention which the oxygenbinding protein is covalently linked to albumin via an optional linker, wherein different functional domain(s) are deleted.

[0061] FIG. 6 describes examples of chimeric protein of the present invention which the oxygenbinding protein is covalently linked to albumin via an optional linker, wherein multiple functional domains of albumin are deleted.

[0062] FIG. 7 describes an expression plasmid that is capable of producing a preferred example chimeric protein - Map of plasmid PB-CMV-HSA-2GS-NGB-2GS-MB-8His-EFS-Puro encoding human serum albumin — human neuroglobin — human myoglobin chimeric protein expression in CHO cells.

[0063] FIG. 8 shows the expression of five chimeric proteins of the present invention by bacterial E. coli, identification by the SDS-PAGE gel and western blot using anti-HSA antibody following purification.

[0064] FIG. 9 shows an example of protein purification of the present invention. A) purification of the human neuroglobin-human serum albumin chimeric protein (“NACP” or “Ngb-HSA” by Immobilized Metal Affinity Chromatography (IMAC) in two elute fractions: aggregate (left peak) and soluble (right peak) forms; and B). SDS-PAGE gel (Coomassie blue staining of proteins) shows both the aggregate and soluble elutes of purified NACP protein have a single band at its predicted molar mass (~84kDa). NACP with SUMO tag (m.w. ~98 kDa) is shown as control.

[0065] FIG. 10 describes visual inspection of oxygen binding to NACP. Iron containing heme group (hemin) added to soluble NACP or “Ngb-HSA” in HEPES buffer (color change from clear to red); Hemin and Hb are extracted and purified from human blood sample

[0066] FIG. 11 describes oxygen binding assays of NACP or “Ngb-HSA”. Spectra of Hb, NACP, Hemin, HSA (equal molar concentration) in oxy- & deoxygenated states.

[0067] FIG. 12 describes oxygen dissociation assays of NACP over 2 hours of deoxygenation.

[0068] FIG. 13 describes oxygen dissociation curves of chimeric proteins and lyophilized human hemoglobin (Lyo Hb).

[0069] FIG. 14 describes spectra signature of lyophilized human Hemoglobin (Lyo Hb) and chimeric proteins in oxy state.

[0070] FIG. 15 describes HSA-Ngb-Mb-His and HSA-Ngb-Mb-Cygb-His chimeric proteins successfully expressed by mammalian cells (HEK 293 and CHO), using standard SDS-PAGE electrophoresis and Western blot (anti-HSA antibodies).

[0071] FIG. 16 describes spectra signature of HSA-Ngb-Mb-His chimeric protein & fresh whole blood (fWB) in oxy- / deoxy-states.

[0072] FIG. 17 describes oxygen equilibrium curves - fresh whole blood (fWB), HSA-Ngb-Mb- His protein, and lyophilized human Hb.

[0073] FIG. 18 describes preferred examples of chimeric protein of the present invention wherein two oxygen-binding proteins (human cytoglobin, human hemoglobin subunit, or myoglobin, or neuroglobin) are covalently linked to human serum albumin via optional linkers at its C-terminaL

[0074] FIG. 19 describes preferred examples of chimeric protein of the present invention wherein three oxygen-binding proteins (human cytoglobin, human hemoglobin subunit, or myoglobin, or neuroglobin) are covalently linked to human serum albumin via optional linkers at its C-terminal.

[0075] FIG. 20 describes preferred examples of chimeric protein of the present invention comprising one albumin, preferably HSA, covalently linked to three oxygen-binding proteins, preferably human myoglobin, neuroglobin or cytoglobin via optional linkers at C-terminal or N- terminal or both.DETAILED DESCRIPTION OF THE INVENTION

[0076] The present invention describes engineered or recombinant chimeric proteins comprising albumin or truncated albumin or albumin variant covalently linked to one or more oxygen binding protein (s), their truncated version, fragments or variants. The present invention further describescompositions comprising such chimeric proteins, applications for the compositions, methods of use, and methods of preparation.

[0077] The invention encompasses eliciting or preventing a biological response which may comprise administering to a subject in need thereof an effective amount of any one of the non- naturally occurring, recombinant or engineered protein(s) or any one of the nucleic acids encoding such non-naturally occurring, recombinant or engineered protein(s) of the present invention, including nucleic acids that may have at least 80% or 85% or 90% or 95% homology or identity with a nucleotide encoding the sequence of the non-naturally occurring recombinant or engineered protein(s) of the invention. The subject may be a mammal, advantageously a primate, advantageously a human.

[0078] As used herein, albumin can be from different species, including animals and humans. Within the present application, albumin is sometimes referred to as ALB or Alb. One preferred example is human serum albumin (“HSA” or “Hsa”), one example of which is shown in Table 3. Another preferrable example is bovine albumin. Albumin gene sequences can be obtained at the National Center for Biotechnology Information (NCBI) database. It is well known that albumin gene sequences may vary from species to species, from person to person. As shown in the NCBI database, different albumin sequences with slight variations can be obtained. As used herein, albumin shall include one or more such variations, including point mutations, addition and deletion.

[0079] Furthermore, additional variations or variants, such as mutations, deletions and additions may be added or introduced into an albumin gene sequence. Accordingly, as used herein, albumin shall include any sequence that is substantially similar to the albumin sequence described herein, and further has identical or has at least 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% sequence homology.

[0080] As used herein, “oxygen-binding protein” refers to a protein whose primary function to bind and / or transport oxygen. Examples of an oxygen binding protein include, without limitation, hemoglobin, myoglobin, neuroglobin and cytoglobin, preferably human hemoglobin, myoglobin, neuroglobin and cytoglobin. Sequences of such oxygen binding protein can be found at the NCBI database, examples of which are shown in Table 3. One of the preferred oxygen binding proteins is myoglobin and neuroglobin. It should be noted that, as used herein, hemoglobin is sometimesreferred to as “HB or Hb”. It should be further noted that, when incorporated into the chimeric proteins of the present invention, hemoglobin shall refer to a subunit of hemoglobin, not a tetramer or dimer. Depending on its source, hemoglobin subunit could be “HBA1”, “HBA2”, “HBB”, “HBG1”, “HBG2”, “HBZ”, or HBE1”. Myoglobin is referred to as “Mb” or “MB”, neuroglobin is referred to as “NGB” or “Ngb” and cytoglobin is referred to as “Cyb” or “CYGB” or “Cygb”.

[0081] One preferable example of an oxygen binding protein is hemoglobin, preferably primate or human hemoglobin. Examples of human hemoglobin can be found at the NCBI database, and one example is shown in Table 3. Similarly, hemoglobin sequence may differ by individuals with certain variations. As used herein, hemoglobin shall include one or more such variations, including point mutations, addition and deletion.

[0082] Oxygen-binding proteins, such as hemoglobin, can be from different species, including animals and humans. One preferable example is human hemoglobin. Thus, as used herein, hemoglobin includes different sequences that have certain sequence variations or variants, such as mutations, deletions and additions, as long as its oxygen carrier function is substantially maintained. Accordingly, as used herein, hemoglobin shall include any sequence that has substantially similar oxygen carrying ability as human hemoglobin and further has a sequence identical or have at least 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% sequence homology as that of a human hemoglobin.

[0083] Table 3 provides amino acid sequence examples of human serum albumin and different oxygen-binding proteins. Some of the sequences contain an optional glycine linker (GGGGS)2. However, it should be noted that the scope of the present invention should not be limited to these examples only.Table 3

[0084] Table 4 provides examples of nucleic acid sequence encoding albumin, different oxygenbinding proteins, and (GGGGS)n (SEQ ID NO: 63) optional linkers (SEQ ID NO: 42-53). However, it should be noted that the scope of the present invention should not be limited to these examples only.Table 4

[0085] Table 5 provides amino acid sequences of some additional preferred examples of the chimeric proteins of the present invention (SEQ ID NO:54-62). However, it should be noted that the scope of the present invention should not be limited to these examples.Table 5

[0086] As used herein, the term “linker” shall refer to a peptide fragment serving to connect the protein moieties (Chen et al, 2013). There are many types of linkers, such as flexible, rigid,cleavable linkers, etc. One preferable example of the present invention is a flexible linker of small and hydrophilic amino acids which may be optimal to preserve chimeric protein’s structure and function(s). Examples of such linker include, but are not limited to, peptide repeat of (GGGGS)n (SEQ ID NO: 63) or (G)n where G is glycine, S is serine, n is preferably betweenl-4. It should be noted that the use of linker may be optional. Albumin or its fragments may be covalently linked to an oxygen- binding protein or truncated version of such protein directly using the bond -C-C-.

[0087] It should be noted that many preferred examples as shown in Table 2 and Table 5 contain one or more glycine linker GGGGS which is underlined. These linkers may be optional and may be deleted. The examples of Table 5 should include chimeric proteins with or without such linkers. Alternative linker(s) may be used to replace such glycine linker.

[0088] It should be further noted that oxygen-binding protein may be altered to increase or decrease its oxygen binding function. For example, His64 or H with its surrounding sequence of SEDLKKHGCTV in human myoglobin moiety may be replaced via site mutagenesis to Gly, Vai, Phe, Met, or Arg. His64 or H with the surrounding sequence of SPEFLDHIRKV in human neuroglobin may be replaced via site mutagenesis to Leu, Vai or Gin; and His64 or H with its surrounding sequence of SPQLRKHACRV in human cytoglobin to Ala, Phe or Trp. Accordingly, the present invention shall further comprise a chimeric protein having one or more of the site mutations.

[0089] As used herein, “effective amount” as used in treatment encompasses, without limitation, an amount that can ameliorate, reverse, mitigate, or prevent a symptom or sign of a medical condition or disorder. Unless dictated otherwise, explicitly or otherwise, an “effective amount” is not limited to a minimal amount sufficient to ameliorate a condition, or to an amount that results in an optimal or a maximal amelioration of the condition.

[0090] The concentration of the chimeric proteins of the present invention, their dosage and the rate of administration should be adjusted to a subject’s individual requirements. The dose required may depend on the size of the subject, the severity of trauma or illness or condition. In case of infusion, measures of adequacy of circulating volume and normal colloid oncotic and osmotic pressure should be used to determine the dose required. Infusion rate and volume need to be adapted according to clinical conditions, most notably in the elderly or in the pediatric population.

[0091] For a normal human subject, human serum albumin protein has a plasma concentration range of 35-50g / L and regulates blood oncotic and osmotic pressure. One embodiment of the present invention is to administer the chimeric proteins so that the plasma concentration in a human subject is the range from Ig / L to 200g / L, more preferably lOg / L to lOOg / L, or 20g / L to lOOg / L, or 30 g / L to 75g / L or even more preferred 36g / L to 60g / L in final plasma concentration so as to achieve similar osmotic pressure.

[0092] The invention pertains to the identification, design, synthesis and isolation of chimeric proteins disclosed herein as well as nucleic acids encoding the same. The present invention also relates to homologues, derivatives and variants of the sequences of a chimeric protein herein and nucleic acids encoding the same, wherein it is preferred that the homologue, derivative or variant have at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98% or at least 99% homology or identity with the sequence of the chimeric proteins or nucleic acids encoding the same. It is noted that within this specification, homology to sequences of the mutant proteins and nucleic acids encoding the same refers to the homology of the homologue, derivative or variant to the binding site of the mutant proteins and nucleic acids encoding the same.

[0093] The invention further relates to nucleic acid sequences expressing the chimeric protein disclosed herein, or homologues, variants or derivatives thereof. One skilled in the art will know, recognize and understand techniques used to create such. Additionally, one skilled in the art will be able to incorporate such a nucleic acid sequence into an appropriate vector, allowing for production of the amino acid sequence of mutant proteins and nucleic acids encoding the same or a homologue, variant or derivative thereof.

[0094] Where used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms might enjoy in the art. The term “isolated” “engineered” or “non-naturally occurring” is used herein to indicate that the isolated moiety (e.g. peptide or compound) exists in a physical milieu distinct from that in which it occurs in nature. For example, the isolated peptide may be substantially isolated with respect to the complex cellular milieu in which it naturally occurs. The absolute level of purity is not critical, and those skilled in the art may readily determine appropriate levels of purity according to the use to which the peptide is to be put. The term “isolating” when used a step in a process is to be interpreted accordingly.

[0095] In many circumstances, the isolated moiety will form part of a composition (for example a more or less crude extract containing many other molecules and substances), buffer system, matrix or excipient, which may for example contain other components (including proteins, such as albumin).

[0096] In other circumstances, the isolated moiety may be purified to essential homogeneity, for example as determined by polyacrylamide gel electrophoresis (PAGE) or column chromatography (for example high performance liquid chromatography [HPLC] or mass spectrometry). In preferred embodiments, the isolated peptide or nucleic acid of the invention is essentially the sole peptide or nucleic acid in a given composition.

[0097] In an advantageous embodiment, a tag may be utilized for purification or biotinylation. The tag for purification may be a His tag. In another embodiment, the tag for biotinylation may be an Avi-tag. Other tags are contemplated for purification; however, purification may be accomplished without a tag. In another embodiment, antibody (such as, not limited to, a broadly neutralizing antibody) affinity columns are contemplated. In another embodiment, lectin columns are contemplated.

[0098] The term “pharmaceutical composition” is used herein to define a solid or liquid composition in a form, concentration and level of purity suitable for administration to a patient (e.g. a human patient) upon which administration it may elicit the desired physiological changes. A “conservative amino acid change” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g. lysine, arginine and histidine), acidic side chains (e.g. aspartic acid and glutamic acid), noncharged amino acids or polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine and cysteine), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan), beta-branched side chains (e.g. threonine, valine and isoleucine), and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan and histidine).

[0099] The terms “protein”, “peptide”, “polypeptide”, and “amino acid sequence” are used interchangeably herein to refer to polymers of amino acid residues of any length. The polymer may be linear or branched, it may comprise modified amino acids or amino acid analogs, and it may be interrupted by chemical moieties other than amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfidebond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling or bioactive component.

[0100] As used herein the terms “nucleotide sequences” and “nucleic acid sequences” refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences, including, without limitation, messenger RNA (mRNA), DNA / RNA hybrids, or synthetic nucleic acids. The nucleic acid may be single-stranded, or partially or completely double-stranded (duplex). Duplex nucleic acids may be homoduplex or heteroduplex.

[0101] As used herein the term “transgene” may be used to refer to “recombinant” nucleotide sequences that may be derived from any of the nucleotide sequences encoding the proteins of the present invention. The term “recombinant” means a nucleotide sequence that has been manipulated “by man” and which does not occur in nature or is linked to another nucleotide sequence or found in a different arrangement in nature. It is understood that manipulated “by man” means manipulated by some artificial means, including by use of machines, codon optimization, restriction enzymes, etc.

[0102] For example, in one embodiment the nucleotide sequences may be mutated such that the activity of the encoded proteins in vivo is abrogated. In another embodiment the nucleotide sequences may be codon optimized, for example the codons may be optimized for human use. In preferred embodiments the nucleotide sequences of the invention are both mutated to abrogate the normal in vivo function of the encoded proteins, and codon optimized for human use.

[0103] As regards codon optimization, the nucleic acid molecules of the invention have a nucleotide sequence that encodes the chimeric proteins of the invention and may be designed to employ codons that are used in the genes of the subject in which the chimeric protein is produced. In a preferred embodiment, the codons used are “humanized” codons, i.e., the codons are those that appear frequently in highly expressed human genes (Andre et al., J. Virol. 72: 1497-1503, 1 98) instead of those codons that are frequently used during protein expression. Such codon usage provides for efficient expression of the chimeric proteins of the present invention. Any suitable method of codon optimization may be used. Such methods, and the selection of such methods, are well known to those of skill in the art. In addition, there are several companies that will optimize codons of sequences, such as Geneart (geneart.com). Thus, the nucleotide sequences of the invention may readily be codon optimized.

[0104] The invention further encompasses nucleotide sequences encoding functionally and / or equivalent variants and derivatives of the chimeric protein of the invention and functionally equivalent fragments thereof. These functionally equivalent variants, derivatives, and fragments display the ability to retain identical or substantially similar activity. For instance, changes in a DNA sequence that do not change the encoded amino acid sequence, as well as those that result in conservative substitutions of amino acid residues, one or a few amino acid deletions or additions, and substitution of amino acid residues by amino acid analogs are those which will not significantly affect properties of the encoded polypeptide.

[0105] Conservative amino acid substitutions are glycine / alanine; valine / isoleucine / leucine; asparagine / glutamine; aspartic acid / glutamic acid; serine / threonine / methionine; lysine / arginine; and phenylalanine / tyrosine / tryptophan. In one embodiment, the variants have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology or identity to the sequences of the present invention.

[0106] For the purposes of the present invention, sequence identity or homology is determined by comparing the sequences when aligned to maximize overlap and identity while minimizing sequence gaps. Sequence identity may be determined by using any of a number of mathematical algorithms. A nonlimiting example of a mathematical algorithm used for comparison of two sequences is the algorithm of Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1990; 87: 2264-2268, modified as in Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1993;90: 5873-5877.

[0107] Another example of a mathematical algorithm used for comparison of sequences is the algorithm of Myers & Miller, CABIOS 1988;4: 11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 may be used. Yet another useful algorithm for identifying regions of local sequence similarity and alignment is the FASTA algorithm as described in Pearson & Lipman, Proc. Natl. Acad. Sci. USA 1988; 85: 2444-2448.

[0108] Advantageous for use according to the present invention is the WU-BLAST (Washington University BLAST) version 2.0 software. WU-BLAST version 2.0 executable programs for several UNIX platforms may be downloaded from ftp: / / blast.wustl.edu / blast / executables. Thisprogram is based on WU-BLAST version 1.4, which in turn is based on the public domain NCBL BLAST version 1.4 (Altschul & Gish, 1996, Local alignment statistics, Doolittle ed., Methods in Enzymology 266: 460-480; Altschul et al., Journal of Molecular Biology 1990, 215: 403-410; Gish & States, 1993, Nature Genetics 3: 266-272; and Karlin & Altschul, 1993, Proc. Natl. Acad. Sci. USA 90: 5873-5877; all of which are incorporated by reference herein).

[0109] The various recombinant nucleotide sequences and chimeric proteins of the present invention can be made using standard recombinant DNA and cloning techniques. Such techniques are well known to those of skill in the art. See for example, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et al. 1989).

[0110] The nucleotide sequences of the present invention may be inserted into “vectors.” The term “vector” is widely used and understood by those of skill in the art, and as used herein the term “vector” is used consistent with its meaning to those of skill in the art. For example, the term “vector” is commonly used by those skilled in the art to refer to a vehicle that allows or facilitates the transfer of nucleic acid molecules from one environment to another or that allows or facilitates the manipulation of a nucleic acid molecule.

[0111] Any vector that allows expression of the chimeric proteins of the present invention may be used in accordance with the present invention. In certain embodiments, the chimeric proteins of the present invention may be used in vitro (such as using cell-free expression systems) and / or in cultured cells grown in vitro in order to produce the desired proteins. For such applications, any vector that allows expression of the proteins in vitro and / or in cultured cells may be used.

[0112] Non-limiting examples of suitable cells in culture include bacteria cells, yeast cells, insect cells, plant cells, and mammalian cells, such as E. coli, S. Cerevisiae, P. pastoris, G. gallus, S. frugiperda, and CHO cells.

[0113] For the nucleic acids of the present invention to be expressed, the protein coding sequence should be “operably linked” to regulatory or nucleic acid control sequences that direct transcription and translation of the protein. As used herein, a coding sequence and a nucleic acid control sequence or promoter are said to be “operably linked” when they are covalently linked in such a way as to place the expression or transcription and / or translation of the coding sequence under the influence or control of the nucleic acid control sequence. The “nucleic acid control sequence” may be any nucleic acid element, such as, but not limited to promoters, enhancers, internal ribosomeentry site (IRES), introns, and other elements described herein that direct the expression of a nucleic acid sequence or coding sequence that is operably linked thereto.

[0114] The vectors used in accordance with the present invention should typically be chosen such that they contain a suitable gene regulatory region, such as a promoter or intergenic region, such that the chimeric proteins of the invention may be expressed.

[0115] Any suitable vector may be used depending on the application. For example, plasmids, viral vectors, bacterial vectors, protozoal vectors, insect vectors, baculovirus expression vectors, yeast vectors, mammalian cell vectors, and the like, may be used. Suitable vectors may be selected by the skilled artisan taking into consideration the characteristics of the vector and the requirements for expressing the immunogens under the identified circumstances.

[0116] Some embodiments of the present invention are the use of viral vectors. Viral expression vectors are well known to those skilled in the art and include, for example, viruses such as adenoviruses, adeno-associated viruses (AAV), alphaviruses, herpesviruses, retroviruses and poxviruses, including avipox viruses, attenuated poxviruses, vaccinia viruses, and particularly, the modified vaccinia Ankara virus (MVA; ATCC Accession No. VR-1566). Such viruses, when used as expression vectors are innately non-pathogenic in the selected subjects such as humans or have been modified to render them non-pathogenic in the selected subjects. For example, replicationdefective adenoviruses and alphaviruses are well known and may be used as gene delivery vectors.

[0117] The nucleotide sequences and vectors of the invention may be delivered to cells, for example if the aim is to express the chimeric protein described herein in cells to produce proteins in cells, any suitable transfections, transformation, or gene delivery methods may be used. Such methods are well known by those skilled in the art, and one of skill in the art would readily be able to select a suitable method depending on the nature of the nucleotide sequences, vectors, and cell types used. For example, transfection, transformation, microinjection, infection, electroporation, lipofection, or liposome-mediated delivery could be used. Expression of the chimeric proteins may be carried out in any suitable type of host cells, such as bacterial cells, yeast, insect cells, and mammalian cells. The chimeric proteins of the invention may also be expressed using in vitro transcription / translation systems. All such methods are well known by those skilled in the art.

[0118] Alternatively, methods which are well known to those skilled in the art may be used to construct expression vectors containing nucleic acid molecules that encode the polypeptide or homologs or derivatives thereof under appropriate transcriptional / translational control signals, forexpression. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination / genetic recombination. See, for example, the techniques described in Maniatis et al., 1989.

[0119] The compounds or compositions may be administered orally, subcutaneously or parenterally including intravenous, intraarterial, intramuscular, intraperitoneally, and intranasal administration as well as intrathecal and infusion techniques. Intramuscular is preferred, but other routes can be used such as subcutaneous or application to mucosal surfaces in the nose or mouth. In an advantageous embodiment, the administration is intravenous. The dosage is measured either in volume or in the effective amount of the chimeric protein described herein.

[0120] It is noted that humans may require higher dosages than mice or other experimental animals to elicit an effective physiological response. The doses may be single doses or multiple doses over a period of time, or continuous infusion. Thus, one may scale up from animal experiments, e.g., rats, mice, and the like, to humans, by technologies from this disclosure and documents cited herein and the knowledge in the art, without undue experimentation.

[0121] When administering a therapeutic of the present invention parenterally, it will generally be formulated in a unit dosage injectable or infusion form (solution, suspension, emulsion). The pharmaceutical formulations suitable for injection or infusion include sterile aqueous solutions or dispersions and sterile powders for reconstitution into sterile injectable or infusion solutions or dispersions. The carrier may be a solvent or dispersing medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. A carrier may be present independently of an adjuvant. For example, the chimeric protein of the present invention can be solved in crystalloid saline, lactated Ringer or other solutions. The solution can be isotonic (0.9% saline) or hypertonic (0.45%) with different concentrations of fusion proteins, 5% or higher.

[0122] Additionally, various additives that enhance the stability, sterility, and isotonicity of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, may be added. Prevention of the action of microorganisms may be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, sorbic acid, and the like. In many cases, it will be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like. According to the present invention, however, any vehicle, diluent, or additive used would have to be compatible with the chimeric proteins of the present invention.

[0123] One advantageous example of the present invention is a dried sterilized form that is suitable for storage and transportation. For example, the chimeric proteins of the present invention may be formulated with specific additives to maintain its stability during drying and storage, typically achieved through a process called freeze-drying (lyophilization) where the protein solution is frozen and then the water is removed by sublimation under vacuum. Thus, another embodiment of the present invention is a lyophilized protein composition comprising the chimeric protein(s) of the present invention.

[0124] Such lyophilized form may be reconstituted at or close to the site of administration with saline, preferably 0.001 to 50 wt.% solution in phosphate buffered saline, diluent or other pharmaceutically acceptable solvent. This is specifically advantageous to natural blood which has limited shelf life and is difficult to store and transport.

[0125] Sterile injectable or infusion solutions may be prepared by incorporating the compounds utilized in practicing the present invention in the required amount of the appropriate buffered solution with various amounts of the other ingredients, as desired.A pharmacological formulation of the present invention, e.g., which may comprise an effective amount of at least one chimeric protein of the present invention, may be administered to a subject in an injectable formulation containing any compatible carrier, such as various vehicles, adjuvants, additives, and diluents; or the chimeric proteins of the present invention may be administered parenterally to the patient in the form of or polymer matrices, liposomes, and microspheres.

[0126] In one embodiment, a formulation of the present invention may be administered initially and thereafter maintained by further administration. For instance, a formulation of the invention may be administered in one type of composition and thereafter further administered in a different or the same type of composition. For example, a formulation of the invention may be administered by intravenous injections or continued infusion to bring blood levels to a suitable level. The patient's levels are then maintained by subsequent administration of injection or infusion.

[0127] Examples of composition include liquid preparations for orifice, e.g., oral, nasal, anal, vaginal, peroral, intragastric, mucosal (e.g., prelingual, alveolar, gingival, olfactory or respiratory mucosa) etc., administration such as suspensions, syrups orandxirs; and preparations for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration), such as sterile suspensions or emulsions. Such compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose orthe like. The compositions may also be lyophilized. The compositions may contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as “REMINGTON'S PHARMACEUTICAL SCIENCE”, 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.

[0128] Compositions of the invention can be conveniently provided as liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions or viscous compositions which may be buffered to a selected pH. Compositions of the invention may contain pharmaceutically acceptable flavors and / or colors for rendering them more appealing, especially if they are administered orally. The viscous compositions may be in the form of gels, lotions, ointments, creams and the like (e.g., for transdermal administration) and will typically contain a sufficient amount of a thickening agent so that the viscosity is from about 2,500 to 6,500 cps, although more viscous compositions, even up to 10,000 cps may be employed. Viscous compositions have a viscosity preferably of 2,500 to 5,000 cps, since above that range they become more difficult to administer. However, above that range, the compositions may approach solid or gelatin forms, which are then easily administered as a swallowed pill for oral ingestion.

[0129] Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection, infusion, or orally. Viscous compositions, on the other hand, may be formulated within the appropriate viscosity range to provide longer contact periods with mucosa, such as the lining of the stomach or nasal mucosa.

[0130] Obviously, the choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage form (e.g., whether the composition is to be formulated into a solution, a suspension, gel or another liquid form), or solid dosage form (e.g., whether the composition is to be formulated into a pill, tablet, capsule, caplet, time release form or liquid-filled form).

[0131] Solutions, suspensions, and gels normally contain a major amount of water (preferably purified water) in addition to the active compound. Minor amounts of other ingredients such as pH adjusters (e.g., a base such as sodium hydroxide), emulsifiers or dispersing agents, buffering agents, preservatives, wetting agents, jelling agents (e.g., methylcellulose), colors and / or flavorsmay also be present. The compositions may be isotonic, i.e. , it may have the same osmotic pressure as blood and lacrimal fluid.

[0132] The desired isotonicity of the compositions of this invention may be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride is preferred particularly for buffers containing sodium ions. Viscosity of the compositions may be maintained at the selected level using a pharmaceutically acceptable thickening agent. Methylcellulose is preferred because it is readily and economically available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The preferred concentration of the thickener will depend upon the agent selected. The important point is to use an amount that will achieve the selected viscosity. Viscous compositions are normally prepared from solutions by the addition of such thickening agents.

[0133] A pharmaceutically acceptable preservative may be employed to increase the shelf-life of the compositions. Benzyl alcohol may be suitable, although a variety of preservatives including, for example, parabens, thimerosal, chlorobutanol, or benzalkonium chloride may also be employed. A suitable concentration of the preservative will be from 0.02% to 2% based on the total weight although there may be appreciable variation depending upon the agent selected.

[0134] Those skilled in the art will recognize that the components of the compositions should be selected to be chemically inert with respect to the active compound. This will present no problem to those skilled in chemical and pharmaceutical principles, or problems may be readily avoided by reference to standard texts or by simple experiments (not involving undue experimentation), from this disclosure and the documents cited herein.

[0135] The compositions of this invention are prepared by mixing the ingredients following generally accepted procedures. For example, the selected components may be simply mixed in a blender, or other standard device to produce a concentrated mixture which may then be adjusted to the final concentration and viscosity by the addition of water or thickening agent and possibly a buffer to control pH or an additional solute to control tonicity. Generally, the pH may be from about 3 to 8.5. Compositions may be administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular patient, and the composition form used for administration (e.g., solidvs. liquid). Dosages for humans or other mammals may be determined without undue experimentation by the skilled artisan, from this disclosure, the documents cited herein, and the knowledge in the art.

[0136] Suitable regimes for initial administration and further doses or for sequential administrations also are variable, and may include an initial administration followed by subsequent administrations; but nonetheless, may be ascertained by the skilled artisan, from this disclosure, the documents cited herein, and the knowledge in the art.

[0137] An effective amount of a chimeric protein described herein may be given in one dose but is not restricted to one dose. Thus, the administration can be two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more, administrations of the vaccine. Where there is more than one administration of the chimeric protein, the administrations can be spaced by time intervals of 1 minute, 2 minutes, 3, 4, 5, 6, 7, 8, 9, 10, or more minutes, by intervals of about 1 hour, 2 hours, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, and so on. In the context of hours, the term “about” means plus or minus any time interval within 30 minutes. The administrations can also be spaced by time intervals of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, and combinations thereof. The invention is not limited to dosing intervals that are spaced equally in time, but encompass doses at non-equal intervals, such as a schedule consisting of administration at 1 day, 4 days, 7 days, and 25 days.

[0138] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined in the appended claims.

[0139] The present invention will be further illustrated in the following Examples which are given for illustration purposes only and are not intended to limit the invention in any way.

[0140] Example 1Construct of Expression Plasmid Encoding Chimeric ProteinExpression plasmid construction based on the sequences of the present invention were carried out using standard recombinant DNA and cloning techniques. Such techniques are well known to those of skill in the art. See for example, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et al. 1989). A total of six plasmids were constructed and the plasmid sequenceencoding for a chimeric protein are shown in Table 1 SEQ ID No 1-6. As designed, these plasmids were for chimeric protein expression in bacteria E. coli (SEQ ID No 1-5) and mammalian CHO cells (SEQ ID No 6). Table 1 SEQ ID No. 4 describes an example of chimeric protein containing human serum albumin covalently linked to human neuroglobin via a glycine linker at C-terminal.

[0141] Example 2Chimeric Protein Expression in BacteriaThe plasmids, as constructed according to Example 1, were then transformed into E. coli BL21 (DE3) cell line. SUMO fusion technology and His tag were used to optimize protein synthesis, cleavage, purification and quantification using routine techniques. See for example, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et al. 1989). Overall, the expression level of the chimeric proteins by E. coli varied and large precipitates were observed in the exclusion body. Follow-up protein denaturing and refolding were required. As shown in Figure 8, five chimeric proteins of the present invention expressed by bacteria E. coli were identified by the SDS-PAGE gel and western blot using anti-HSA antibody following purification. Arrows indicate chimeric proteins expressed; E refers to eluted chimeric protein with SUMO tag (m.w. ~14 kDa). U refers to Ubl-specific protease 1 treated chimeric protein (removal of SUMO tag). As used herein, HSA refers to human serum protein, Hba2-HSA refers to human hemoglobin oc2-human serum albumin, Hb|32-HSA refers to hemoglobin p2-human serum albumin, Mb-HSA refers to human myoglobin- human serum albumin, Ngb-HSA refers to human neuroglobin-human serum albumin, Cygb-HSA refers to human cytoglobin- human serum albumin chimeric proteins.

[0142] Example 3Chimeric protein design and in silico modelingThe molecular weight of the purified chimeric proteins was calculated and predicted, ranging from 80 to 90 kDa (>60 kDa renal clearance limit) as shown in Table 6. To predict the water solubility of the chimeric proteins of Example 2, a protein pool (Prot-pl) developed by Zurich University (www.propi.ch) was used to calculate the pl of natural globin and chimeric proteins. As shown in Table 6, the chimeric proteins of the present invention are predicted to have slightly acidic pl compared to their natural forms and should be water soluble and have renal retention rates (circulation residence time) similar to that of natural albumin.Table 6. The chimeric proteins & isoelectric points

[0143] Example 4Protein Purification for NACP (Ngb-HSA) and MeasurementThe IMAC purification was earned out using HisPur Purification Kit (Thermo Scientific) according to the manufacturer specifications. The chimeric proteins are tagged with SUMO protein (cleaved post expression) and histidine for purification. Following SUMO Tag removal, Neuroglobin-Albumin Chimeric Protein (“NACP”) was purified by immobilized metal affinity chromatography. There were two major elute fractions observed: the aggregate corresponding to misfolded protein and the soluble, correct folded NACP (Figure 9A). In SDS-PAGE, both the aggregae and soluble elutes of purified NACP protein showed a single band at its predicted molar mass (~84kDa) (Figure 9B). NACP with SUMO tag (m.w. ~98 kDa) is shown as control.

[0144] Example 5Visual Inspection of Oxygen Binding to NACPPrior to in vitro function testing, a solution containing hemin group was added to NACP. The addition of the heme group turned NACP in HEPE buffer from clear to red color as shown in Figure 10, indicating integration of hemin in correctly folded NACP and potential oxygen binding to the NACP.

[0145] Example 6Oxygen Binding AssaysTo measure oxygen dissociation, NACP (with hemin added), human lyophilized hemoglobin (Human Hb), hemin, and albumin samples were each diluted to the same molar concentration (5 pM) in HEPES buffer in a 96-well plate. After oxygenation, the samples were deoxygenated with compressed N2 for 2 hours in HEPES buffer without 2,3-DPG. Oxygen dissociation assay (ODA) was set at the spectral signatures of Hb (Soret bands: 400-450 nm and Q bands: 500-600 nm) to measure the level of oxy-protein during deoxygenation. The method developed by former GlobalBlood Therapeutics team (Patel 2018) for a novel screening assay based on the spectral changes observed during deoxygenation of oxy-Hb was used for the testing. The assay can detect and quantify oxygenated Hb and NACP in real time on a 96-well plate and results were validated by measurement on standard Hemox Analyzer instrument. Absorption peak was detected in the 400- 415nm range for NACP, corresponding to the reported signature spectrum for free form of neuroglobin at -410 nm (Figure 11 A & B). Human Hb also showed an oxygen binding absorption in the 400-415nm range as positive control. Albumin and Hemin group showed no oxygen binding as negative controls. Data confirmed that NACP as produced in Example 2 was in monomeric form and was shown to bind oxygen.

[0146] Example 7Oxygen Dissociation AssayOxygen dissociation assay was performed using NACP. After oxygenation, the samples were deoxygenated with compressed N2 for 2 hours in HEPES buffer without 2,3-DPG. Oxygen dissociation assay (ODA) used the spectral signatures of Hb (Soret bands: 400-450 nm and Q bands: 500-600 nm) to measure the level of oxy-protein during deoxygenation (Patel 2018). The assay used the spectral signatures of individual chimeric protein (Soret bands: 400-450 nm and Q bands: 500-600 nm) to measure the level of oxygen binding. Purified proteins (3 LIM in various buffers) were incubated for 1 hour under ambient air at 37°C 96-well, optically transparent polystyrene plates. After incubation, the samples were deoxygenated with gaseous dry N2 for 2 hours at 37°C. During deoxygenation process, spectral measurements (350-700 nm, with a spectral resolution of 1-5 nm) were obtained every 6 minutes to assess the oxygen binding level over time. In between each measurement, the plate was shaken for 1 minute at 300 rpm. Data analysis was performed using Excel’s LINEST function on the wavelengths ranging from 380 to 700 nm. The protein spectrum at time zero was used as the reference for 100% oxy-globin-HSAs, while the average spectra of protein with 15 mM sodium dithionite (Merck Millipore, USA), a reducing agent, was used as the reference for 100% deoxygenated state. The results were then expressed as % of oxygen binding. The results were shown in Figure 12A, indicating that the oxygen binding to NACP declined over time. The rate of dissociation for NACP was slower than that of hemoglobin as shown in Figure 12B, suggesting that NACP may have a higher binding affinity to oxygen than Hemoglobin.

[0147] Example 8Oxygen Dissociation Curves of Chimeric Proteins and lyophilized hemoglobin Following 30 min oxygenation in ambient air, the samples were deoxygenated with compressed N2 gas for 2 hours. Rate of deoxygenation was analyzed and plotted against time TO to quantitate the level of deoxygenation vs. Lyo Hb. HbP2-HSA, Ngb-HSA, Mb-HSA, and Cygb-HSA exhibited slower rates of oxygen dissociation relative to Lyo Hb as shown in Figure 13. All experiments were conducted using Bio-Tek microplate reader (Agilent Technologies, USA) at 37°C in HEPES buffer (pH 7.4) without addition of 2,3-DPG.

[0148] Example 9Spectra Signature of chimeric proteins vs Lyo Hb in oxy stateChimeric protein samples (Ngb-, Mb-, Cygb-HSA, Lyo Hb, 10 mM each) in a 96-well plate were incubated in ambient air (oxygenation) for an hour and spectra (A. 390-680nm) were measured. Fully oxygenated protein of Lyo Hb, Hba2- & Hb[32-HSA (10 mM each) exhibit strong Soret band absorption near 410 nm & weak Q bands around 530 / 570 nm as shown in Figure 14 A. the UV-visible absorption spectra for. Ngb-HSA, Mb-HSA, and Cygb-HSA displayed characteristic oxygen-binding spectra, with prominent Soret absorption near 410 nm and subdued Q bands and minor shift in Soret bands as shown in Figure 14B. All experiments were conducted using Bio- Tek microplate reader (Agilent Technologies, USA) at 37°C in HEPES buffer (pH 7.4) without addition of 2,3-DPG.

[0149] Example 10Expression and Purification of HSA-Ngb-Mb-His and HSA-Ngb-Mb-Cygb-His Chimeric Proteins in Mammalian CellsTo avoid protein misfolding and bacterial endotoxin contamination, the expression plasmids were constructed to produce the chimeric proteins in mammalian HEK293 and Chinese Hamster Ovary (CHO) cell. Example of such expression plasmids was shown in Figure 7. Proteins were produced, harvested and purified using routine techniques. Such techniques were well known to those of skill in the art. See for example, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et al. 1989). To generate the chimeric protein producing cell lines, CHO-S cells were transfected with a proprietary expression plasmid containing cDNA encoding selected chimeric protein sequence (HSA-Ngb-Mb-His or HSA-Ngb-Mb-Cygb-His) and with a plasmid with cDNA encoding a2,3-sialyltransferase (ST3GAL_IV). After transfection, cells were pooled and seeded at 1.0 xlO6viable cells per ml culture medium in T-flasks. Cells were incubated for48 h and seeded in 96-well plates at 300 to 3000 viable cells / well in culture medium supplemented with Zeocin (Invitrogen) and Blasticidin (Invitrogen) (selection medium). The cells were incubated for -1 week. Visible cell clones were then transferred into 24- well plates containing 2 ml fresh selection medium and screened for the protein levels (HSA ELISA). Best producing clones were selected, cultured in 6-well plates and expanded into 25 ml T-flasks (4- day culture). Subsequently, they adapted to culture under shaking conditions and cultured for 7 days while cell density, viability and production are monitored at various intervals. In addition, sialylation of the chimeric proteins produced by different clones was analyzed using lectin-based ELISAs. Finally, two cell lines producing the chimeric proteins with respectively low and high sialylation were selected.

[0150] Stable cell lines were re-seeded in large tissue culture plates (100mm) and incubated at 37 °C in serum free media for up to sevemdays. The culture supernatant containing the expressed chimeric protein was collected and concentrated by Amicon Ultra 15 filter (lOOkDa cut-off) in a centrifuge.

[0151] To purify a His-tagged protein using Ni2+-NTA (nickel-charged) beads or a column, the concentrated cell supernatant containing the expressed protein was first equilibrated with a binding buffer (His60 Ni buffer set, TaKaRa) to minimize nonspecific binding. The supernatant was then incubated with Ni-NTA resin or loaded onto a pre-equilibrated column, allowing the His-tag to chelate nickel ions on the resin. After binding, the resin was washed with buffer containing a moderate concentration of imidazole (typically 20-40 mM) to remove weakly bound contaminants while retaining the His-tagged protein. Finally, the target protein was eluted using a higher concentration of imidazole (100-500 mM). The eluted fractions were collected, analyzed (e.g., by SDS-PAGE), and prepared for downstream applications.

[0152] Using standard SDS-PAGE electrophoresis and Western blot (anti-HSA antibodies), chimeric proteins (with His tag peptide) contained in culture supernatant were separated, labeled, and validated (protein bands corresponding to their predicted molecular mass: HSA-Ngb-Mb-His -lOOkDA, HSA-Ngb-Mb-Cygb-His, ~130kDa) as shown in Figure 15A. The presence of a single band in both cell lines confirms expression of the full-length chimeric protein and indicates protein stability.

[0153] Example 11Visual Inspection of Oxygen Binding to HSA-Ngb-Mb-His chimeric protein expressed by mammalian cellsCell cultural supernatant containing secreted proteins in serum-free media was concentrated by Amicon Ultra 15 filter (lOOkDa cut-off). No heme / hemin group was added. The concentrated cell culture supernatant containing HSA-Ngb-Mb-His chimeric protein appeared clear with a distinct blood-red hue without addition of exogenous heme / hemin as shown in Figure 15B, implying soluble chimeric protein and natural oxygen binding with heme de novo synthesized by both HEK293 and CHO cells.

[0154] Example 12Spectra Signature Assaying of HSA-Ngb-Mb-His Chimeric Protein as Compared to Human Whole BloodTo assess the oxy- and deoxy-state of the chimeric protein, UV / Vis absorbance was measured at spectra wavelength bands (Soret: 400-450 nm; Q: 500-600 nm). HSA-Ngb-Mb-His chimeric protein (100 pM) and fresh whole blood (10 pM) in HEPES buffer (pH 7.4) were incubated at 37°C under ambient air for 1 hour in a BioTek Synergy microplate reader (Agilent Technologies, Winooski, VT). Additional samples were incubated either in low pH (6.75) HEPES buffer or addition of reducing agent-sodium dithionite (15 mM). Full spectral scans (380-690 nm) at 1 nm resolution were collected and spectra band.

[0155] As Figure 16A shown, UV-Vis absorption spectra of HSA-Ngb-Mb-His protein (lOOuM), fully oxygenated protein showing broad and humped Soret band absorption near 414 nm & pronounced single Q band around 562 nm. This is distinct from the absorption spectra for fWB (10 uM) with signature profile of a sharp Soret band at 416 nm and twin Q bands around 542 & 578 nm. Following addition of sodium dithionite (15mM), HSA-Ngb-Mb-His protein’s spectra 414 nm band shifts to 428 nm and flattening of 562 nm Q band, indicates fully deoxy state. Incubation in pH 6.75 buffer attenuated Q band absorption of the chimeric protein, implying oxygen unloading under acidic conditions. As in Figure 16B shown, absorption spectra for fWB (10 uM) exhibited signature Soret band at 418nm and twin Q bands around 542 & 578 nm. Signature spectra shift - 418 nm Soret band to 430 nm and 542 nm / 578 nm twin Q bands to 560nm- following addition of reducing agent demonstrated deoxygenation. Low pH 6.75 induced partial deoxygenation, reducing absorption without major band shifts.

[0156] Example 13Oxygen Equilibrium Assay for HSA-Ngb-Mb-His chimeric protein as Compared to Human Whole Blood and Lyophilized Human HemoglobinHSA-Ngb-Mb-His (25 pM), fWB (10 pM), and Lyo Hb (10 pM) were incubated for 45 minutes at 37°C with in HEPES buffer (pH7.4). The samples were then oxygenated with compressed air for 10 minutes in TCS Hemox Blood Analyzer. After oxygenation, the samples were deoxygenated with compressed N2 gas and OECs were collected during deoxygenation. From the OECs, p50 values were derived using TCS Hemox Analysis Software. The HSA-Ngb-Mb-His chimeric protein displayed a distinct OEC profile, characterized by efficient oxygen uptake and mid-range binding affinity (P50 = 9.56 mmHg) in between fWB P50 = 17.59 mmHg and Lyo Hb P50 = 5.25 mmHg, as shown in Figure 17.

[0157] Example 14Antishock and Safety Study in Rat Extremity Trauma ModelMale Sprague-Dawley rats (~13 weeks old) are anesthetized, intubated, and catheterized to enable controlled hemorrhage and continuous monitoring of mean arterial pressure (MAP) and heart rate (HR). Following induction of soft tissue injury and fibular fracture, blood is withdrawn to maintain MAP at 55 mmHg for a 45-minute shock phase.

[0158] After shock induction, animals are randomized into four intravenous treatment groups (n = 7-8 per group; ~35 total): saline, 25% albumin, 25% equivalent osmolar HSA-Ngb-Mb-His protein, or fWB. All treatments are administered at matched volumes (0.3ml / 100g of body weight). Systemic hemodynamics (MAP, HR), renal functions (RBF, urinary flow, GFR, sodium excretion), and skeletal muscle capillary flow and oxygen partial pressure are recorded over a two- hour post- treatment period. Blood gases (pO2, sO2, pCO2), creatinine (Cr), blood urea nitrogen (BUN), glucose, and electrolytes are measured at baseline, after 45 min of shock, and at 30 min intervals post- treatment using an i-STAT Handheld Blood Analyzer (Abbott Point of Care, Inc, Princeton, NJ).

[0159] To assess renal blood flow (RBF), a perivascular ultrasonic transit-time flow probe (0.7PSB; Transonic Systems, Inc., Ithaca, NY) is positioned around the left renal artery prior to surgical closure of the abdominal incision. Renal vascular resistance (RVR) will be calculated based on MAP / RBF. Glomerular filtration rate (GFR) is determined using the fluoresceinisothiocyanate (FITC)-sinistrin method (10 mg / kg; NIC-Kidney, Mannheim Pharma & Diagnostics, Germany). Skeletal muscle capillary perfusion and the tissue oxygen partial pressureare measured using an Oxylite NX PO2 / flow bare fiber sensor inserted into the right hindlimb (OxyFlo; Oxford Optronix Ltd, United Kingdom). The entire protocol is independently replicated three times (total n ~ 100). Data from all replicates are pooled and analyzed for statistically significant differences to evaluate therapeutic efficacy and safety across treatment groups.

[0160] Example 15Purification and lyophilization of chimeric proteinsThe stable expression CHO-S cells are cultured in ProCHO4 medium. The cell suspension is then incubated at 3LC for 6 days in a shaking incubator. To obtain a desired production volume of IL, the procedure is scaled up. Size exclusion chromatography is used for protein purification from the culture supernatant. Subsequent purification by protein separation columns will also be used. Fractions containing chimeric protein are pooled and dialyzed in phosphate buffer, assessed for purity and protein content by running a protein gel, a BCA protein assay (Thermo Scientific) and the HSA ELISA, aliquoted and stored at -80°C until further analysis. Using standard SDS-PAGE electrophoresis and Western blot (anti-HSA antibodies), chimeric proteins are separated, labeled, and validated (protein bands corresponding to their predicted molecular mass). Purified chimeric protein will be formulated in saline or phosphate- buffered solutions, adjusted to 5% or 25% albumin-equivalent osmotic concentrations, for use in in vitro and in vivo testing.

[0161] Lyophilization (freeze-dry) is the standard process for protein products (in dry powder form) transport and long-term storage. But lyophilization may cause chimeric protein to unfold, denature, or autoxidize, which can alter its function. To prevent oxidative damage, carbon monoxide (CO) gas (in a fume hood) is added into protein solution as standard practice for oxygen carriers (1 hour at room temperature). Saccharide stabilizers (sucrose or trehalose) are also added to the solution before freezing overnight at -80°C. The frozen samples are lyophilized at 0.002 mbar for 2 hours at room temperature. The lyophilized chimeric protein powder will be stored in a sterile, light- shielding, vacuum- sealed, airtight Mylar bag for stability studies.

[0162] Example 16Blood Transfusion Assay in Rat Hemorrhage ModelRats are anesthetized with 1.5% isoflurane and 2 L / min of O2 and secured in the supine position on a metal board. They are given 0.5 mg / kg of buprenorphine subcutaneously before surgery. They are positioned with the tail toward the surgeon after the carotid artery catheter was placed. Following anesthesia, a midline laparotomy is performed to expose the abdominal cavity and theleft lobe of the liver. Three triangle-shaped filter papers (pre-weighed ~0.035 g each) are inserted into the abdominal cavity. A 3.0xl.5-cm section of the edge of left liver lobe is transected with sharp scissors to cause liver injury. Following the procedure, the abdominal skin is securely closed using wound clips (AutoClip kit, Fine Science Tools) to avoid leakage of blood. Rats are monitored for the duration of the experiment (15, 60-minute time points). At the end of the experiment, wound clips are removed to reopen the abdominal cavity and collect filter papers. A fourth piece of preweighed filter paper is used to absorb any remaining blood in the abdominal cavity. Blood loss is determined by weighing the blood-soaked filter papers and is expressed in microliters per gram. Within one hour of the surgery and laceration, animals will be randomized to receive either the current chimeric proteins or controls in a two-by-two factorial design: four groups receiving the chimeric proteins intravenously at a dose of either lx or 2x of albumin amount calculated from blood volume lost; two control groups receiving plasma or saline at a volume equivalent to the lost plasma volume measured.

[0163] LDH is widely used as a general indicator of acute or chronic tissue damage and is considered an inflammatory marker (Sepulveda 2013). In situations such as shock, severe injury, or heart failure, elevated blood lactate levels are commonly used as a marker to indicate tissue hypoxia, as they suggest compromised oxygen delivery to tissues (Valenza 2003). In this proposed study, both LDH and Lactate level in the blood will be measured over time as biomarkers for tissue damage and proxies for O2 delivery and distribution. For short-term readout, a small amount (50 ml) of blood from retroorbital veins will be drawn 1 hour following injection of chimeric proteins or controls. Both LDH and the lactate levels in the blood are collected and tested as short-term readouts. In follow-up measurement, both LDH and lactate levels will be tested on day 1, 3, 5 post trauma / therapeutic injection. On day 5, the end point, all the animals are harvested for histopathology analysis to measure the level of major tissue damage in brains, hearts and livers. The rat bleeding model is expected to provide crucial data for the efficacy and safety of the chimeric protein of the present protein.

[0164] Example 17Human Blood Transfusion TestFor clinical studies in human, the chimeric protein is tested first in patients undergoing acute normovolemic hemodilution (ANH) cardiac surgery, which requires the removal of blood from a patient before surgery. The patient's circulating blood volume is currently maintained with infusionof crystalloid or colloid solutions. The ANH cardiac surgery study can enroll elective cardiac surgery patients with cardiopulmonary bypass (CPB) who will be randomized 1:1 to the chimeric protein vs Crystalloid or SOC groups (Monaco 2024). The primary endpoint can be all-cause 30- day mortality, and secondary outcomes are acute kidney injury, ischemic complications and reduction of RBC transfusion during hospital stay.

[0165] The chimeric protein is also tested in autoimmune hemolytic anemia (AIHA) which is a life-threatening condition and high unmet medical need. AIHA patients often suffer severe anemia when their own immune system attacks their RBC and require frequent allogenic blood transfusions. Blood transfusion becomes challenging in late stage AIHA patients when hemolytic transfusion reactions (HTRs) develop rendering it ineffective and more toxic.References:Alayash Al, Mechanism of Toxicity and Modulation of Hemoglobin-based Oxygen Carriers, Shock, October 2019Alayash Al, Oxidation reactions of cellular and acellular hemoglobins: Implications for human health, Frontiers in Medical Technology, 28 November 2022Benitez Cardenas AS, Samuel PP, Olson JS, Current challenges in the development of acellular hemoglobin oxygen carriers by protein engineering. Shock 52 (Suppl. 1):28- 40, 2019.Buehler PW, D’ Agnillo F, Schaer DJ: Hemoglobin-based oxygen carriers: From mechanisms of toxicity and clearance to rational drug design. Trends Mol Med 16(10):447^457, 2010.Burmester, T. et al. A vertebrate globin expressed in the brain, Nature, Vol 407, September 2000Burmester T & Hankeln T, What is the function of neuroglobin, J. Exp Biol., 2009, 212, 1423- 1428Chambers JA, et al. Stop the bleed: a US military installation and model for implementation of a rapid hemorrhage, Military Medicine, 2019 184:67-71Chen, XY, et al. Fusion Protein Linkers: Property, Design and Functionality, Adv. Drug Deliv.Rev. 2013 Oct 15:65 1357-1369Dou Y, et al. Myoglobin as a model system for designing heme protein based blood substitutes,Biophysical Chemistry, 2002, 127-148Fernandez-Moure J, Maisha N, Lavik EB, The Chemistry of Lyophilized Blood Products, Bioconjug Chem. 2018 Jul 18 ;29(7):2150-2160Garry DJ, Kanatous SB, Mammen PP, Emerging roles for Myoglobin in the heart, 2003, vol 13, issue 3, 111-116Gupta AS, Hemoglobin-based oxygen carriers: current state-of-the-art and novel molecules, Shock, October 2019Hill-Pryor, C. et al. Hemoglobin-Based Oxygen Carriers (HBOC)-What the Next Generation Holds: When Red Blood Cells Are Not An Option, Shock, volume 52, 2019Jahr, JS, Lowery, DR, et al. Blood Substitutes and Oxygen Therapeutics: A Review, Anesthesia & Analgesia, January 2021Jahr, JS Blood substitutes: Basic science, translational studies and clinical trials, Frontiers in Medical Technology, August 2022Jahr, JS and Williams JP, Blood Component Requirements and Erythrocyte Transfusion and Mortality Related to Hemoglobin Deficit in Phase III Trial of Hemoglobin-Based Oxygen Carrier: HBOC-201, American Journal of Therapy, May-Jun 2022Larsen MT et al, Albumin-based drug delivery: harnessing nature to cure disease, Molecular and Cellular Therapies, 20164:3Mathai, C. et al. Emerging perspectives on cytoglobin, beyond NO dioxygenase and peroxidase, Redox Biology, January 2020Monaco F, et al. Acute normovolemic hemodilution in cardiac surgery: rational design of a multicenter randomized trial, Contemporary Clinical Trials 143 (2024) 107605Pires IS, Berthiaume F, Palmer AF, Engineering Therapeutics to Detoxify Hemoglobin, Heme, and Iron, Annual Review of Biomedical Engineering, 2023 25: 1-21Patel, MP, et al. Development and validation of oxygen dissociation assay, a screening platform for discovering, and charactering hemoglobin-oxygen affinity modifiers, Drug Design, Development and Therapy, 2018:12 1599-1607Pesce, A. et al. Human Brain Neuroglobin Structure Reveals a Distinct Mode of Controlling Oxygen Affinity, Structure, vol 11, September 2003Raoufinia, R., et al. Overview of Albumin and its purification methods, Adv. Pharm. Bull., 2016 Dec 22: 495-507Reeder, BJ, Insights into the function of cytoglobin, Biochemical Society Transactions, September 2023Sepulveda J, Challenges in Routine Clinical Chemistry Analysis: Proteins and Enzymes.Editor(s): A. Dasgupta, J. L. Sepulveda, Chapter 9, Accurate Results in the Clinical Laboratory, Elsevier, 2013:131-148.Tam MF, et al. Autoxidation and oxygen binding properties of recombinant hemoglobins with substitutions at the aVal-62 or bVal-67 position of the distal Heme Pocket, J. Biol. Chem., 2013 Jul 18Taverna, M, et al. Specific antioxidant properties of human serum albumin Annals of Intensive Care, 2013, 3:4Tsujino H, et al. Disulfide bonds regulate binding of exogenous ligand to human cytoglobin, J. of Inorganic Biochem., Vol 135, June 2014, 20-27Varnado, CL, et al. Development of Recombinant Hemoglobin-Based Oxygen Carriers, Antioxidants & Redox Signaling, volume 18, 2013, 2314-2328Valenza F, et al. Lactate as a marker of energy failure in critically ill patients: hypothesis, Critical Care, 2005 Sep 28;9(6) :588-593Vanek T. and Kohli, A. Biochemistry, Myoglobin July 17, 2023Wright TJ and Davis RW, Myoglobin oxygen affinity in aquatic and terrestrial birds and mammals, J Exp Biol. 2015 Jul; 218 (pt 14):2180-9Xiang L, et al., Can polyethylene glycol-20k replace albumin for prehospital treatment of hemorrhagic shock when full resuscitation is unavailable? Shock 59 (5), 725-733. doi: 10.1097 / SHK.0000000000002099Zaleski, A. There will be blood, Science News, July 2024* *Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.

[0166] All documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

Claims

1. WHAT IS CLAIMED IS:

1. An engineered chimeric protein comprising a human serum albumin, and any fragment thereof, covalently linked to at least one oxygen binding protein.

2. The chimeric protein of claim 1, wherein the oxygen binding protein is selected from a group consisting of hemoglobin, myoglobin, neuroglobin and cytoglobin.3 The chimeric protein of claim 1, wherein the oxygen binding protein is hemoglobin.4 The chimeric protein of claim 1, wherein the protein has a sequence selected from SEQ ID Nos. 7-29 and 54-62 or their homologs.5 A pharmaceutical composition comprising a human serum albumin, and any fragment thereof, covalently linked to at least one oxygen binding protein and a pharmaceutically acceptable carrier.6 The chimeric protein of claim 5, wherein the oxygen binding protein is selected from a group consisting of hemoglobin, myoglobin, neuroglobin and cytoglobin.7 The chimeric protein of claim 5, wherein the oxygen binding protein is hemoglobin.8 The chimeric protein of claim 5, wherein the protein has a sequence selected from SEQ ID Nos. 7-29 and 54-62 or their homologs.9 A method of treating or preventing a disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of a chimeric protein comprising a human serum albumin, and any fragment thereof, covalently linked to at least one oxygen-binding protein.10 The method of claim 9, wherein the disease or condition is selected from a group consisting of ischemic stroke, blood transfusion, hemorrhage shock, hemoglobinopathy, hemolytic disorders, sepsis, trauma, anemia, dialysis, traumatic brain injury, myocardial infarction, pulmonary hypertension, cystic fibrosis, hypovolemia, ascites, hypoalbuminemia including from burns, acute nephrosis, acute respiratory distress syndrome, cardiopulmonary bypass, preservation of organ transplant.11 The method of claim 9, wherein the disease or condition is blood transfusion.12 The chimeric protein of claim 9, wherein the oxygen binding protein is selected from a group consisting of hemoglobin, myoglobin, neuroglobin and cytoglobin.

13. The chimeric protein of claim 9, wherein the oxygen binding protein is hemoglobin.

14. The chimeric protein of claim 9, wherein the protein has a sequence selected from SEQ ID Nos. 7-29 and 54-62 or their homologs.

15. A composition for treating, preventing or delaying aging or age-related symptoms or conditions in a subject in need thereof, comprising an effective amount of the chimeric protein of Claims 1-4.

16. A method of treating, preventing or delaying aging or age-related symptoms or conditions, comprising administering to a subject in need thereof an effective amount of the chimeric protein of Claims 1-4.

17. A composition for promoting longevity in a subject in need thereof, wherein the composition comprises an effective amount of at least one chimeric protein of Claims 1-4.

18. A method for promoting longevity in a subject comprising administering to the subject in need thereof an effective amount of the chimeric protein of Claims 1-4.

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