Compositions and methods for innate immune modulators

Abstract

The present disclosure provides compositions comprising a modified bactericidal permeability increasing protein (BPI) and pharmaceutical compositions thereof. The modified BPIs and pharmaceutical compositions thereof may be used in a variety of therapeutic methods for the treatment of immune related diseases or disorders.

Description

COMPOSITIONS AND METHODS FOR INNATE IMMUNEMODULATORSRELATED APPLICATIONS

[0001] This application claims the priority and benefits of US. Provisional Application No. 63 / 612,941, filed December 20, 2023, the contents of which are incorporated herein by reference in their entirety.SEQUENCE LISTING

[0002] The Sequence Listing XML associated with this application is provided in XML file format and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing XML is RSTH_001_01WO_SeqList_ST26.xml. The XML file is 53,130 bytes in size, was created on December 20, 2024, and is being submitted electronically via USPTO Patent Center.TECHNICAL FIELD

[0003] The present disclosure provides innate immune modulators and pharmaceutical compositions comprising the same, and methods of use thereof.BACKGROUND

[0004] The present disclosure provides compositions comprising innate immune modulators, such as modified bactericidal permeability increasing protein (BPI). BPI is a protein found in granules of mammalian polymorphonuclear leukocytes and has potent bactericidal activity against a broad range of gram-negative bacteria. The bactericidal activity of BPI is highly specific; it is generally non-toxic for gram-positive bacteria, fungi, and eukaryotic cells.

[0005] Increasingly, gram-negative bacteria pose major risks and cause a significant disease burden. Gram-negative bacteria cause infections including pneumonia, bloodstream infections, wound or surgical site infections, and meningitis. The frequency and severity of these infections can be higher for subjects with certain diseases or disorders including neutropenia, thrombocytopenia, and graft-versus-host disease, as well as after hematopoietic cell transplantation. Antimicrobial resistance among gram-negative bacteria is an urgent global problem, and gram-negative bacteria have the potential for rapid spread of resistance mechanisms. Treatment options for gram-negative bacteria are increasingly limited. In particular, thedevelopment of multidrug-resistant strains, which are resistant to three or more classes of antimicrobials, or extensively drug-resistant strains, which are resistant to all but one or two classes of antimicrobials, is a cause of major concern.

[0006] Additionally, gram-negative bacteria pose as hurdles to hematopoietic recovery after exposure to radiation and / or chemotherapy and are involved in immune disorders such as graft- versus host disease (GvHD), immune thrombocytopenia, Myelodysplastic Syndromes, Fanconi Anemia, acute radiation syndrome, gastrointestinal-acute radiation syndrome, drug resistant gramnegative infections, and sepsis.

[0007] Accordingly, compositions comprising innate immune modulators such as BPI and modified forms thereof are needed.SUMMARY

[0008] In various embodiments, the present disclosure provides a recombinant polypeptide comprising (a) an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1-5; and (b) an Fc fragment. In some embodiments, the amino acid sequence comprises any one of SEQ ID NOs: 1-5.

[0009] In some embodiments, the Fc fragment is human IgGl. In some embodiments, the Fc fragment comprises an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 6. In some embodiments, the Fc fragment comprises an amino acid sequence comprising SEQ ID NO: 6.

[0010] In some embodiments, the recombinant polypeptide is linked to the Fc fragment by a linker. In some embodiments, the linker is a Gly-Ser rich linker. In some embodiments, the linker is a Gly-Ser linker. In some embodiments, the linker comprises a sequence selected from the group consisting of: GSAGSAAGSGEF (SEQ ID NO: 38), (GGGGS)n (SEQ ID NO: 39), GGGS (SEQ ID NO:40), and GGGGS (SEQ ID NO:39). In some embodiments, the linker is GGGS (SEQ ID NO:40).

[0011] In some embodiments, the amino acid sequence comprises a sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 7- 11. In some embodiments, the amino acid sequence comprises any one of SEQ ID NOs: 7-11.

[0012] In some embodiments, the recombinant polypeptide comprises (a) amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1-5; and (b) a polyhistidine-tag (His-tag). In some embodiments, the amino acid sequence comprises any one of SEQ ID NOs: 1-5. In some embodiments, the His-tag is at the C-terminus or the N-terminus of the recombinant polypeptide. In some embodiments, the His-tag is at the C-terminus of the recombinant polypeptide. In some embodiments, the His-tag is an 8X- His-tag.

[0013] In some embodiments, the recombinant polypeptide comprises comprising a sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 12-16. In some embodiments, the recombinant polypeptide comprises any one of SEQ ID NOs; 12-16.

[0014] In some embodiments, the recombinant polypeptide comprises a sequence encoding a signal peptide. In some embodiments, the sequence encoding the signal peptide comprises SEQ ID NO: 17 or SEQ ID NO: 18.

[0015] In some embodiments, the recombinant polypeptide comprises the sequence of any one of SEQ ID NOs: 19 - 34 or 43 - 44.

[0016] In some embodiments, the present disclosure provides a composition comprising any one or more of the recombinant polypeptides disclosed herein. In some embodiments, the recombinant polypeptide is covalently attached to polyethylene glycol (PEG). In some embodiments wherein the recombinant polypeptide is covalently attached to PEG, the recombinant polypeptide comprises an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1-5. In some embodiments wherein the recombinant polypeptide is covalently attached to PEG, the recombinant polypeptide comprises any one of SEQ ID NOs: 1-5. In some embodiments, the PEG is covalently attached to the His- tag of the recombinant polypeptide. In some embodiments, the PEG is PEG-40.

[0017] In some embodiments, the present disclosure provides a pharmaceutical composition comprising any one or more of the recombinant polypeptides or compositions disclosed herein, and a pharmaceutically acceptable excipient.

[0018] In some embodiments, the present disclosure provides an isolated nucleic acid molecule comprising a nucleic acid sequence encoding any one or more of the recombinant polypeptides disclosed herein.

[0019] In some embodiments, the isolated nucleic acid molecule comprises a heterologous promoter. In some embodiments, the heterologous promoter is selected from the group consisting of: CMV, CMV-IE, CMVZEFlalpha, and SV40. In some embodiments, the heterologous promoter is a CMV promoter.

[0020] In some embodiments, the present disclosure provides a vector comprising any one or more of the isolated nucleic acid molecules described herein. In some embodiments, the vector is a non-viral vector. In some embodiments, the non-viral vector is a transposon vector. In some embodiments, the transposon vector is a PiggyBac transposon vector.

[0021] In some embodiments, the present disclosure provides a cell comprising any one or more of the isolated nucleic acid molecules and / or vectors described herein.

[0022] In some embodiments, the present disclosure provides a kit comprising any one or more of the isolated nucleic acid molecules and / or vectors described herein, and optionally instructions for use.

[0023] In some embodiments, the present disclosure provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject any one or more of the recombinant polypeptides, compositions, or pharmaceutical compositions disclosed herein. In some embodiments, the disease or disorder is selected from the group consisting of: extensively drug resistant gram-negative infections, graft vs host disease (GvHD), Fanconi amenia, chemotherapy induced neutropenia, chemotherapy induced thrombocytopenia, myeloablative radiation therapy induced neutropenia, and myeloablative radiation therapy induced thrombocytopenia.

[0024] In some embodiments, the present disclosure provides a method of promoting hematopoietic recovery in a subject in need thereof, comprising administering to the subject any one or more of the recombinant polypeptides, compositions, or pharmaceutical compositions disclosed herein. In some embodiments, the disease or disorder is graft-versus-host disease (GvHD), immune thrombocytopenia, myelodysplastic syndrome (MDS), Fanconi anemia, or acute radiation syndrome (ARS).BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1 provides a schematic of an exemplary vector comprising a CMV promoter and a selectable marker.

[0026] FIG. 2 provides a schematic showing the structure of RT-1042, an exemplary BPI-Fc fusion molecule.

[0027] FIG. 3 provides a graph showing the survival curve of three groups of mice that underwent total body irradiation (TBI), with one group untreated (6Gy), one group receiving vehicle control (Vehicle), and one group receiving RT-1042.

[0028] FIG. 4 provides a schematic showing how conditioning regimens, such as TBI, result in the destruction of the intestinal epithelia and the release of endotoxin from Gram Negative bacteria, thereby triggering a proinflammatory cascade.

[0029] FIG. 5 provides a Kaplan-Meier plot showing survival of mice that underwent TBI and allogenic cell transplantation and treated with vehicle, RT-1042 (a BPI-Fc fusion molecule), RT- 1274 (BPI-PEG), or RT-127 (BPI-His).

[0030] FIG. 6 provides a graph showing the neutralization of LPS by RT-1042 or RT-127, calculated as % activity remaining.DETAILED DESCRIPTIONOverview

[0031] The present disclosure provides compositions comprising a modified bactericidal permeability increasing protein (BPI) and methods for using modified BPI for treatment of immune-related diseases or disorders. Modified BPI can function as an innate immune modulator. Innate immune modulators are substances that can stimulate or suppress the innate immune system, which is the body's first line of defense. Innate immunity is the body's ability to quickly respond to and destroy foreign substances, such as bacteria or viruses, that enter the body. Innate immunity and adaptive immunity are highly interconnected. Therefore, without being held to theory, innate immune modulators can play a crucial role in overcoming immune tolerance and avoid resistance being developed, for example, against immune checkpoint inhibitors in cancer therapy. However, native BPI has a very short half-life, which renders it unsuitable as a therapeutic. Compositions of the present disclosure comprise modified BPI, and may provide improved characteristics such as, but not limited to, improved pharmacokinetics (PK) and improved therapeutic effects.Bactericidal Permeability Increasing Protein (BPI) Polypeptides

[0032] The present disclosure provides recombinant polypeptides comprising bactericidal permeability increasing protein (BPI) polypeptides.

[0033] In some embodiments, the recombinant polypeptides comprise a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 1-5. In some embodiments, the recombinant polypeptides comprise any one of SEQ ID NOs: 1-5. In some embodiments, the recombinant polypeptides consists of any one of SEQ ID NOs: 1-5.Exemplary BPI sequences

[0034] VNPGVVVRISQKGLDYASQQGTAALQKELKRIKIPDYSDSFKIKHLGKGHYSFYS MDIREFQLPSSQISMVPNVGLKFSISNANIKISGKWKAQKRFLKMSGNFDLSIEGMSISADLKLGSNPTSGKPTITASSCSSHINSVHVHISKSKVGWLIQLFHKKIESALRNKMNSQVCE KVTNSVSSELQPYFQTL (SEQ ID NO: 1),

[0035] VNPGVVVRISQKGLDYASQQGTAALQKELKRIKIPDYSDSFKIKHLGKGHYSFYS MDIREFQLPSSQISMVPNVGLKFSISNANIKISGKWKAQKRFLKMSGNFDLSIEGMSISAD LKLGSNPTSGKPTITASSCSSHINSVHVHISKSKVGWLIQLFHKKIESALRNKMNSQVCE KVTNSVSSELQP (SEQ ID NO: 2),

[0036] VNPGVVVRISQKGLDYASQQGTAALQKELKRIKIPDYSDSFKIKHLGKGHYSFYS MDIREFQLPSSQISMVPNVGLKFSISNANIKISGKWKAQKRFLKMSGNFDLSIEGMSISAD LKLGSNPTSGKPTITASSCSSHINSVHVHISKSKVGWLIQLFHKKIESALRNKMNSQVCE KVTNSV (SEQ ID NO: 3),

[0037] VNPGVVVRISQKGLDYASQQGTAALQKELKRIKIPDYSDSFKIKHLGKGHYSFYS MDIREFQLPSSQISMVPNVGLKFSISNANIKISGKWKAQKRFLKMSGNFDLSIEGMSISAD LKLGSNPTSGKPTITASSCSSHINSVHVHISKSKVGWLIQLFHKKIESALRNKMNSQVCE K (SEQ ID NO: 4),

[0038] VNPGVVVRISQKGLDYASQQGTAALQKELKRIKIPDYSDSFKIKHLGKGHYSFYS MDIREFQLPSSQISMVPNVGLKFSISNANIKISGKWKAQKRFLKMSGNFDLSIEGMSISAD LKLGSNPTSGKPTITASSCSSHINSVHVHISKSKVGWLIQLFHKKIESALRNKMNS (SEQ ID NO: 5).

[0039] In some embodiments, the recombinant polypeptides comprise SEQ ID NO: 1. In some embodiments, the recombinant polypeptides comprise SEQ ID NO: 2. In some embodiments, the recombinant polypeptides comprise SEQ ID NO: 3. In some embodiments, the recombinant polypeptides comprise SEQ ID NO: 4. In some embodiments, the recombinant polypeptides comprise SEQ ID NO: 5.

[0040] The present disclosure also provides recombinant polypeptides comprising a BPI polypeptide, wherein the BPI polypeptide is linked to an immunoglobulin Fc fragment and / or a tag. In some embodiments, the BPI polypeptide is a functional fragment of a BPI polypeptide, with or without linkage to an immunoglobulin Fc fragment or tag. In some embodiments, the recombinant polypeptides disclosed herein comprises a functional fragment of a wild type or recombinant BPI polypeptide. In some embodiments, the recombinant polypeptides disclosed herein comprises a functional fragment of a wild type BPI polypeptide. In certain embodiments, the functional fragment of the wild type BPI polypeptide comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% sequence identity to a wild type BPI polypeptide or any one of SEQ ID NOs: 1-5.

[0041] The recombinant polypeptides comprising a recombinant BPI polypeptide or a functional fragment thereof described herein may comprise at least one modification relative to a wild type BPI polypeptide. In some embodiments, the at least one modification comprises a deletion, an insertion, or a substitution of at least one amino acid relative to a wild type BPI polypeptide.

[0042] In some embodiments, the at least one modification comprises a deletion relative to a wild type BPI polypeptide. In some embodiments, the deletion comprises deletion of at least one amino acid residue at the C-terminal, N-terminal, and / or a middle portion of the polypeptide relative to the wild type BPI polypeptide. In some embodiments, the deletion comprises deletion of at least 2 consecutive amino acid residues or at least 2 non-consecutive amino acid residues. In some embodiments, the deletion comprises deletion of at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, or at least 175 consecutive amino acids. In some embodiments, the deletion comprises a truncation, where at least one amino acid is deleted from the N-terminal or C-terminal of the polypeptide.

[0043] In some embodiments, the at least one modification comprises an insertion relative to a wild type BPI polypeptide. In some embodiments, the insertion comprises insertion of at least one amino acid residue at the C-terminal, N-terminal, and / or a middle portion of the polypeptide relative to the wild type BPI polypeptide. In some embodiments, the insertion comprises insertion of at least 2 consecutive amino acid residues or at least 2 non-consecutive amino acid residues. In some embodiments, the insertion comprises insertion of at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, or at least 175 consecutive amino acids. In some embodiments, at least one amino acid is inserted at the N-terminal or C-terminal of the recombinant polypeptide.

[0044] In some embodiments, the at least one modification comprises an amino acid substitution relative to a wild type BPI polypeptide. In some embodiments, the substitution comprises substitution of at least one amino acid residue at the C-terminal, N-terminal, and / or a middle portion of the protein relative to the wild type BPI polypeptide. In some embodiments, the substitution comprises substitution of at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, or at least 175 amino acids.

[0045] In some embodiments, the recombinant polypeptides described herein comprises an Fc fragment, also referred to as an Fc domain or Fc. The Fc fragment may be derived from any known class of immunoglobulin. Non-limiting examples include a wild-type or modified IgGl, IgG2, IgG3, IgG4 or other isotype, e.g., wild-type or modified human or murine IgGl, human or murine IgG2, human or murine IgG3, human IgG4, human IgG4Pro (comprising a mutation in core hinge region that prevents the formation of IgG4 half molecules), human or murine IgA, human or murine IgE, human or murine IgM, or human or murine IgM. In some embodiments, the Fc domain is derived from IgG. In some embodiments, the Fc domain is derived from a human IgG. In some embodiments, the Fc domain is derived from a human IgGl. In some embodiments, the Fc domain is derived from a murine IgG2a. In some embodiments, all or part of the Fc is humanized.

[0046] In some embodiments, the Fc comprises amino acid substitutions corresponding to L234A, L235A, P329G (LALA-PG) in SEQ ID NO: 6 and / or as described in Lo et al. J. Biol. Chem. 2017; 292, p. 3900-3908. The LALA-PG variant has been shown to eliminate complement binding and fixation as well as Fc-g dependent antibody-dependent cell- mediated cytotoxicity (ADCC) in both murine IgG2a and human IgGl. These LALA-PG substitutions allow a more accurate translation of results generated with an effector-less antibody framework scaffold between mice and primates.

[0047] In some embodiments, the immunoglobulin Fc fragment is derived from SEQ ID NO: 6. In some embodiments, the Fc fragment comprises a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 6. In some embodiments, the Fc fragment comprises SEQ ID NO: 6.

[0048] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP EAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 6)

[0049] The present disclosure also provides recombinant polypeptides comprising a BPI polypeptide wherein the BPI polypeptide is linked to an immunoglobulin Fc fragment via a linker. In some embodiments, the linker is a Gly-Ser rich linker. In some embodiments, the linker is a Gly-Ser linker. In some embodiments, the linker comprises a sequence selected from the groupconsisting of: GSAGSAAGSGEF (SEQ ID NO: 39), (GGGS)n, wherein n = 1 to 20 (SEQ ID NO:40), GGGS (SEQ ID NO: 41), and GGGGS (SEQ ID NO: 42). In some embodiments, the linker comprises (GGGS)n, wherein n = 1 to 20 (SEQ ID NO: 40). In some embodiments, the linker comprises GGGS (SEQ ID NO: 41). In some embodiments, the linker is GGGS (SEQ ID NO:41).

[0050] The present disclosure also provides recombinant polypeptides comprising a BPI polypeptide wherein the BPI polypeptide is linked to an immunoglobulin Fc fragment via a linker. As used herein the term “BPI-Fc” refers to a recombinant polypeptide comprising a BPI polypeptide wherein the BPI polypeptide is linked to an immunoglobulin Fc fragment. In some embodiments, the recombinant BPI-Fc polypeptide disclosed herein comprises a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 7-11. In some embodiments, , the recombinant BPI-Fc polypeptide comprises any one of SEQ ID NOs: 7-11. In some embodiments, the recombinant BPI-Fc polypeptide consists of any one of SEQ ID NOs: 7-11.Exemplary BPI-Fc sequences

[0051] VNPGVVVRISQKGLDYASQQGTAALQKELKRIKIPDYSDSFKIKHLGKGHYSFYS MDIREFQLPSSQISMVPNVGLKFSISNANIKISGKWKAQKRFLKMSGNFDLSIEGMSISAD LKLGSNPTSGKPTITASSCSSHINSVHVHISKSKVGWLIQLFHKKIESALRNKMNSQVCE KVTNSVSSELQPYFQTLGGGSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<ALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7),

[0052] VNPGVVVRISQKGLDYASQQGTAALQKELKRIKIPDYSDSFKIKHLGKGHYSFYS MDIREFQLPSSQISMVPNVGLKFSISNANIKISGKWKAQKRFLKMSGNFDLSIEGMSISAD LKLGSNPTSGKPTITASSCSSHINSVHVHISKSKVGWLIQLFHKKIESALRNKMNSQVCE KVTNSVSSELQPGGGSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<ALPAPIEI<TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 8),

[0053] VNPGVVVRISQKGLDYASQQGTAALQKELKRIKIPDYSDSFKIKHLGKGHYSFYS MDIREFQLPSSQISMVPNVGLKFSISNANIKISGKWKAQKRFLKMSGNFDLSIEGMSISAD LKLGSNPTSGKPTITASSCSSHINSVHVHISKSKVGWLIQLFHKKIESALRNKMNSQVCE KVTNSVGGGSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFP AVLQS SGL YSLS S VVTVPS S SLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 9),

[0054] VNPGVVVRISQKGLDYASQQGTAALQKELKRIKIPDYSDSFKIKHLGKGHYSFYS MDIREFQLPSSQISMVPNVGLKFSISNANIKISGKWKAQKRFLKMSGNFDLSIEGMSISAD LKLGSNPTSGKPTITASSCSSHINSVHVHISKSKVGWLIQLFHKKIESALRNKMNSQVCE KGGGSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 10), VNPGVVVRISQKGLDYASQQGTAALQKELKRIKIPDYSDSFKIKHLGKGHYSFYSMDIR EFQLPSSQISMVPNVGLKFSISNANIKISGKWKAQKRFLKMSGNFDLSIEGMSISADLKL GSNPTSGKPTITASSCSSHINSVHVHISKSKVGWLIQLFHKKIESALRNKMNSGGGSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11).

[0055] In some embodiments, the recombinant polypeptides disclosed herein has a longer cellular half-life than wild-type BPI. For example, the recombinant polypeptides described herein may have a half-life that is at least about 10%, at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% longer than that of a wild-type BPI polypeptide. In some embodiments, the recombinant polypeptides described herein may have a half-life that is about 4 hours, about 12 hours, about 18 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 10 days, or about 10 days longer than that of a wild-type BPI polypeptide.

[0056] The present disclosure also provides recombinant polypeptides comprising a BPI polypeptide wherein the BPI polypeptide is linked to a tag. Non-limiting examples of tags include polyhistidine tags (His-tags), glutathione-S-transferase tags (GST-tags), FLAG tags, streptavidin tags, maltose binding protein (MBP) tags, HA tags, c-Myc tags, and Spot-tags. In some embodiments, the recombinant polypeptides disclosed herein comprise a His-tag. As used herein the term “BPI-His” refers to a recombinant polypeptide comprising a BPI polypeptide linked to a His-tag. Non-limiting examples of His-tags include sequences comprising at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten consecutive histidines. As used herein, the terms “3X His-tag”, “4X His-tag”, “5X His-tag”, “6X His-tag”, “7X His-tag”, “8X His-tag”, “9X His-tag”, and “10X His-tag” refer to a tag with 3, 4, 5, 6, 7, 8, 9, or 10 consecutive histidines, respectively.

[0057] In some embodiments, the recombinant BPI-His polypeptide described herein comprises a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 1-5, additionally comprising a His-tag. In some embodiments, the His-tag is at the C-terminus or the N-terminus of the recombinant polypeptide. In some embodiments, the His-tag is at the C-terminus of the recombinant polypeptide. In some embodiments, the His-tag is an 8X-His-tag.

[0058] In some embodiments, the recombinant BPI-His polypeptide described herein comprises a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 12-16. In some embodiments, the recombinant BPI-His polypeptide comprises any one of SEQ ID NOs: 12-16. In some embodiments, the recombinant BPI-His polypeptide consists of any one of SEQ ID NOs: 12-16.Exemplary BPI-His sequences

[0059] VNPGVVVRISQKGLDYASQQGTAALQKELKRIKIPDYSDSFKIKHLGKGHYSFYS MDIREFQLPSSQISMVPNVGLKFSISNANIKISGKWKAQKRFLKMSGNFDLSIEGMSISADLKLGSNPTSGKPTITASSCSSHINSVHVHISKSKVGWLIQLFHKKIESALRNKMNSQVCE KVTNSVSSELQPYFQTLHHHHHHHH (SEQ ID NO: 12),

[0060] VNPGVVVRISQKGLDYASQQGTAALQKELKRIKIPDYSDSFKIKHLGKGHYSFYS MDIREFQLPSSQISMVPNVGLKFSISNANIKISGKWKAQKRFLKMSGNFDLSIEGMSISAD LKLGSNPTSGKPTITASSCSSHINSVHVHISKSKVGWLIQLFHKKIESALRNKMNSQVCE KVTNSVSSELQPHHHHHHHH (SEQ ID NO: 13),

[0061] VNPGVVVRISQKGLDYASQQGTAALQKELKRIKIPDYSDSFKIKHLGKGHYSFYS MDIREFQLPSSQISMVPNVGLKFSISNANIKISGKWKAQKRFLKMSGNFDLSIEGMSISAD LKLGSNPTSGKPTITASSCSSHINSVHVHISKSKVGWLIQLFHKKIESALRNKMNSQVCE KVTNSVHHHHHHHH (SEQ ID NO: 14),

[0062] VNPGVVVRISQKGLDYASQQGTAALQKELKRIKIPDYSDSFKIKHLGKGHYSFYS MDIREFQLPSSQISMVPNVGLKFSISNANIKISGKWKAQKRFLKMSGNFDLSIEGMSISAD LKLGSNPTSGKPTITASSCSSHINSVHVHISKSKVGWLIQLFHKKIESALRNKMNSQVCE KHHHHHHHH (SEQ ID NO: 15),

[0063] VNPGVVVRISQKGLDYASQQGTAALQKELKRIKIPDYSDSFKIKHLGKGHYSFYS MDIREFQLPSSQISMVPNVGLKFSISNANIKISGKWKAQKRFLKMSGNFDLSIEGMSISAD LKLGSNPTSGKPTITASSCSSHINSVHVHISKSKVGWLIQLFHKKIESALRNKMNSHHHH HHHH (SEQ ID NO: 16).BPI polypeptides with signal sequences

[0064] The present disclosure also provides recombinant polypeptides comprising a BPI polypeptide and a signal peptide. In some embodiments, the signal peptide comprises a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 17 or SEQ ID NO: 18. In some embodiments, the signal peptide comprises SEQ ID NO: 17 or SEQ ID NO: 18. In some embodiments, the signal peptide comprises SEQ ID NO: 17. In some embodiments, the signal peptide comprises SEQ ID NO: 18. In some embodiments, the signal peptide consists of SEQ ID NO: 17 or SEQ ID NO: 18. In some embodiments, the signal peptide consists of SEQ ID NO: 17. In some embodiments, the signal peptide consists of SEQ ID NO: 18.Exemplary signal peptide sequences

[0065] MARGPCNAPRWVSLMVLVAIGTAVTAA (SEQ ID NO: 17).

[0066] MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 18).PEGylation of Modified BPI

[0067] The present disclosure also provides compositions comprising any one of the recombinant polypeptides disclosed herein. In some embodiments, the composition comprises a recombinant polypeptide comprising a sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 1-5, wherein the recombinant polypeptide is covalently attached to polyethylene glycol (PEG). In some embodiments, the composition comprises a recombinant polypeptide comprising the sequence of any one of SEQ ID NOs: 1-5, wherein the recombinant polypeptide is covalently attached to polyethylene glycol (PEG).

[0068] Polymers, and particularly PEG, are highly flexible and soluble and have gained widespread scientific and regulatory acceptance as a chemical modification for therapeutic proteins. For example, PEG attachment (PEGylation) improves PK predominantly by increasing the effective size of a protein. PEGylation can also reduce immunogenicity and aggregation. The modified BPI disclosed herein can be PEGylated to increase biological half-life by reacting the modified BPI or fragment thereof with polyethylene glycol (PEG) or a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the modified BPI or fragment thereof. PEGylation may be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water soluble polymer). PEGs can be attached to proteins at lysines and cysteines via NHS-ester or maleimide activated PEGs, respectively. In some embodiments, the compositions disclosed herein is PEGylated via maleimide chemistry. In some embodiments, the PEG attached to the compositions disclosed herein is PEG-40.

[0069] Several PEGylated protein therapeutics are currently on the market or in late-stage clinical trials. Schering-Plough's PEG-Intron® (peginterferon alfa-2b) and Roche's PEGasys® (peginterferon alfa-2a), both PEGylated variants of interferon-a (IFNa) used to treat hepatitis C, show significantly improved in vivo efficacy relative to the parent molecules.

[0070] However, many PEGylated protein therapeutics can have significantly reduced specific activity relative to the unmodified proteins (see for example Bailon, P. et al. (2001) “Rational design of a potent, long-lasting form of interferon: a 40-kDa-branched polyethylene glycol- conjugated interferon alpha-2a for the treatment of hepatitis C” Bioconjug. Chem. 12, 195-202; and Wang, Y. S. et al. (2002) “Structural and biological characterization of PEGylated recombinant interferon alpha-2b and its therapeutic implications” Adv. Drug Deliv. Rev. 54, 547- 570). Thus, although PEG attachment is generally useful for improving pharmacokinetics, it oftendoes so at the expense of specific activity. As a result, it is necessary to find PEG attachment sites that minimally impact the specific activity of the modified protein.

[0071] In some embodiments of the compositions described herein comprising any one of the recombinant polypeptides disclosed herein and a His-tag, a PEG is covalently linked to the His- tag. In some embodiments, the PEG is covalently linked to a histidine of the His-tag (e.g., as described in Cong, et al. Bioconjugate Chemistry. 2012; 23(2):248-63). In some embodiments, PEG-bis-sulfone undergoes site-specific conjugation to the histidines in the His-tag through bis- alkyation with the nitrogens on the imidazole ring of histidine to create a covalent conjugate. In some embodiments, the resulting BPI-His-PEG is then separated from non-conjugated BPI-His by size exclusion chromatography.Exemplary amino acid sequences

[0072] Exemplary amino acid sequences for recombinant BPI, BPI-His, and BPI-Fc polypeptides are provided in Table 1 below. Unless otherwise indicated, signal peptide sequences are indicated by underlining and linker sequences are indicated in bold.Table 1 : Exemplary amino acid sequences for recombinant polypeptides comprising BPIPharmaceutical Compositions

[0073] The present disclosure also provides pharmaceutical compositions comprising any one of the recombinant polypeptides or compositions disclosed herein. In some embodiments, thepharmaceutical composition additionally comprises a pharmaceutically acceptable diluent, carrier, or excipient. In some embodiments, the pharmaceutical composition additionally comprises a pharmaceutically acceptable excipient.

[0074] Pharmaceutically-acceptable carriers, diluents, and excipients may be any known in the art. In some embodiments, pharmaceutically-acceptable carriers, diluents, and excipients are those that are useful in preparing a formulation that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for mammalian, e.g., human or primate, use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous. Examples of such carriers, diluents and excipients include, but are not limited to, water, saline, Ringer’s solutions, dextrose solution, and 5% human serum albumin. Supplementary active compounds can also be incorporated into the formulations. Solutions or suspensions used for the formulations can include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates; detergents such as Tween 20 to prevent aggregation; and compounds for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. In some embodiments, the pharmaceutical compositions are sterile.

[0075] Pharmaceutical compositions may further include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). In some cases, the composition is sterile and should be fluid such that it can be drawn into a syringe or delivered to a subject from a syringe. In certain embodiments, it is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, e.g., a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, forexample, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the internal compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0076] Sterile solutions can be prepared by incorporating any one or more of the recombinant polypeptides and / or compositions described herein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.Methods of Treatment

[0077] The present disclosure also provides methods of treating a disease or disorder in a subject in need thereof, comprising administering one or more of the recombinant polypeptides, compositions, pharmaceutical compositions, nucleic acids, and / or vectors disclosed herein. The recombinant polypeptides, compositions, pharmaceutical compositions, nucleic acids, and / or vectors described herein are administered in an amount sufficient to improve a symptom of the disorder or disease.

[0078] Symptoms of diseases or disorders described herein can be evaluated using standard methods known to those of skill in the art and may be reduced (e.g., the subject's symptoms may be improved) by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%) compared to symptoms prior to administration of the recombinant polypeptides, compositions, pharmaceutical compositions, nucleic acids, and / or vectors described herein. These effects may occur, for example, within 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, or more, following administration of the recombinant polypeptides, compositions, pharmaceutical compositions, nucleic acids, and / or vectors described herein. The patient may be evaluated 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or more following administration of the recombinant polypeptides, compositions, pharmaceutical compositions, nucleic acids, and / or vectors depending on the dose and route of administration used for treatment.

[0079] In some embodiments, the disease or disorder is selected from the group consisting of: extensively drug resistant gram-negative infections, graft-vs-host disease (GvHD), Fanconi amenia, chemotherapy induced neutropenia, chemotherapy induced thrombocytopenia, myeloablative radiation therapy induced neutropenia, and myeloablative radiation therapy induced thrombocytopenia.

[0080] Various factors, such as certain drugs, chemicals, immune disorders, and exposure to radiation and / or chemotherapy, can lead to bone marrow failure. Bone marrow failure ischaracterized by marrow deficiency evidenced in one or more hematopoietic lineages, including a deficiency in myeloid cells, a deficiency in leukocytes (leukopenia), a deficiency in megakaryocytes (megakaryopenia), a deficiency in platelets (thrombocytopenia), a deficiency in monocytes, a deficiency in lymphocytes (lymphopenia) , a deficiency in erythrocytes (erythropenia), a deficiency in neutrophils (neutropenia), a deficiency in T cells, a deficiency in granulocytes, and / or a deficiency in dendritic cells. In some embodiments, the recombinant polypeptides, compositions, and / or pharmaceutical compositions described herein can be used to promote hematopoietic recovery in a subject with bone marrow failure. In some embodiments, the recombinant polypeptides, compositions, pharmaceutical compositions, nucleic acids, and / or vectors disclosed herein promote hematopoietic recovery in a subject with graft-versus-host disease (GvHD). In some embodiments, the recombinant polypeptides, compositions, pharmaceutical compositions, nucleic acids, and / or vectors disclosed herein promote hematopoietic recovery in a subject with immune thrombocytopenia. In some embodiments, the recombinant polypeptides, compositions, pharmaceutical compositions, nucleic acids, and / or vectors disclosed herein promote hematopoietic recovery in a subject with a myelodysplastic syndrome (MDS). In some embodiments, the recombinant polypeptides, compositions, pharmaceutical compositions, nucleic acids, and / or vectors disclosed herein promote hematopoietic recovery in a subject with Fanconi anemia. In some embodiments, the recombinant polypeptides, compositions, pharmaceutical compositions, nucleic acids, and / or vectors disclosed herein promote hematopoietic recovery in a subject with acute radiation syndrome (ARS).

[0081] In some embodiments, the recombinant polypeptides, compositions, pharmaceutical compositions, nucleic acids, and / or vectors described herein promote recovery in a subject with Gastrointestinal-Acute Radiation Syndrome. In some embodiments, the recombinant polypeptides, compositions, pharmaceutical compositions, nucleic acids, and / or vectors described herein are used for the treatment of multidrug-resistant, extensively drug-resistant, and / or pandrug-resistant Gram-negative infections, such as Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. In some embodiments, the recombinant polypeptides, compositions, pharmaceutical compositions, nucleic acids, and / or vectors described herein are used for the treatment of sepsis.

[0082] The recombinant polypeptides, compositions, pharmaceutical compositions, nucleic acids, and / or vectors described herein may be administered to a subject by any mode of delivery, including, for example, by parenteral injection (e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue), or by rectal, oral (e.g. tablet,spray), vaginal, topical, transdermal (e.g. see WO99 / 27961) or transcutaneous (e.g. see WO02 / 074244 and W002 / 064162), intranasal (e.g. see W003 / 028760), ocular, aural, pulmonary or other mucosal administration and / or inhalation of powder compositions. Multiple doses can be administered by the same or different routes.

[0083] The recombinant polypeptides, compositions, pharmaceutical compositions, nucleic acids, and / or vectors described herein may be administered to a subject once, or more than once. For example, the recombinant polypeptides, compositions, and / or pharmaceutical compositions described herein may be administered one, two, three, four, five, six, seven, eight, nine, ten, or more times. Doses may be administered at any known interval. For example, doses may be administered every six hours, every eight hours, every 12 hours, every 18 hours, daily, every two days, twice weekly, weekly, twice monthly, or monthly.

[0084] The dose and dosage regimen may depend upon a variety of factors readily determined by a physician, such as the nature of the disease or condition, the characteristics of the subject, and the subject's history. In some embodiments, the effective amount of the recombinant polypeptides, compositions, or pharmaceutical compositions described herein is about 10 mg to about 10,000 mg per day. In some embodiments, the effective amount the recombinant polypeptides, compositions, or pharmaceutical compositions is about 100 mg to about 5,000 mg per day. In some embodiments, the effective amount the recombinant polypeptides, compositions, or pharmaceutical compositions is about 1,000 mg to about 3,000 mg per day. In some embodiments, the effective amount the recombinant polypeptides, compositions, or pharmaceutical compositions is about 0.1 mg / kg to about 300 mg / kg mg per day. In some embodiments, the effective amount the recombinant polypeptides, compositions, or pharmaceutical compositions is about 1 mg / kg to about 100 mg / kg mg per day. In some embodiments, the effective amount the recombinant polypeptides, compositions, or pharmaceutical compositions is about 10 mg / kg to about 30 mg / kg.Polynucleotides

[0085] The present disclosure also provides isolated nucleic acid molecules comprising a nucleic acid sequence encoding one or more of the recombinant polypeptides described herein.

[0086] In some embodiments, the isolated nucleic acid disclosed herein encodes one or more of the amino acid sequences of any one of SEQ ID NOs: 1 - 5. In some embodiments, the isolated nucleic acid comprises a sequence with at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 35. In some embodiments, the isolated nucleic acid comprises SEQ ID NO: 35.Exemplary BPI polynucleotide sequenceGTC AAC CCT GGC GTC GTG GTC AGG ATC TCC CAG AAG GGC CTG GAC TAC GCC AGC CAG CAG GGG ACG GCC GCT CTG CAG AAG GAG CTG AAG AGG ATC AAG ATT CCT GAC TAC TCA GAC AGC TTT AAG ATC AAG CAT CTT GGG AAG GGG CAT TAT AGC TTC TAC AGC ATG GAC ATC CGT GAA TTC CAG CTT CCC AGT TCC CAG ATA AGC ATG GTG CCC AAT GTG GGC CTT AAG TTC TCC ATC AGC AAC GCC AAT ATC AAG ATC AGC GGG AAA TGG AAG GCA CAA AAG AGA TTC TTA AAA ATG AGC GGC AAT TTT GAC CTG AGC ATA GAA GGC ATG TCC ATT TCG GCT GAT CTG AAG CTG GGC AGT AAC CCC ACG TCA GGC AAG CCC ACC ATC ACC GCC TCC AGC TGC AGC AGC CAC ATC AAC AGT GTC CAC GTG CAC ATC TCA AAG AGC AAA GTG GGG TGG CTG ATC CAA CTC TTC CAC AAA AAA ATT GAG TCT GCG CTT CGA AAC AAG ATG AAC AGC CAG GTC TGC GAG AAA GTG ACC AAT TCT GTA TCC TCC GAG CTG CAA CCT TAT TTC CAG ACT CTG TAG (SEQ ID NO: 35).

[0087] In some embodiments, the isolated nucleic acid disclosed herein further encodes the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18. In some embodiments, the isolated nucleic acid comprises a sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 36. In some embodiments, the isolated nucleic acid comprises SEQ ID NO: 36.Exemplary signal peptide polynucleotide sequenceATG GCC AGG GGC CCT TGC AAC GCG CCG AGA TGG GTG TCC CTG ATG GTG CTC GTC GCC ATA GGC ACC GCC GTG ACA GCG GCC (SEQ ID NO: 36).

[0088] In some embodiments, the isolated nucleic acid disclosed herein encodes the amino acid sequence of SEQ ID NOs: 6. In some embodiments, the isolated nucleic acid comprises a sequence with at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 37. In some embodiments, the isolated nucleic acid comprises SEQ ID NO: 37.Exemplary IgGl polynucleotide sequence gcgagcaccaaaggcccgagcgtgtttccgctggcgccgagcagcaaaagcaccagcggc ggcaccgcggcgctgggctgcctggtgaaagattattttccggaaccggtgaccgtgagc tggaacagcggcgcgctgaccagcggcgtgcatacctttccggcggtgctgcagagcagc ggcctgtatagcctgagcagcgtggtgaccgtgccgagcagcagcctgggcacccagacc tatatttgcaacgtgaaccataaaccgagcaacaccaaagtggataaaaaagtggaaccg aaaagctgcgataaaacccatacctgcccgccgtgcccggcgccggaagcggcgggcggcccgagcgtgtttctgtttccgccgaaaccgaaagataccctgatgattagccgcaccccg gaagtgacctgcgtggtggtggatgtgagccatgaagatccggaagtgaaatttaactgg tatgtggatggcgtggaagtgcataacgcgaaaaccaaaccgcgcgaagaacagtataac agcacctatcgcgtggtgagcgtgctgaccgtgctgcatcaggattggctgaacggcaaa gaatataaatgcaaagtgagcaacaaagcgctgccggcgccgattgaaaaaaccattagc aaagcgaaaggccagccgcgcgaaccgcaggtgtataccctgccgccgagccgcgatgaa ctgaccaaaaaccaggtgagcctgacctgcctggtgaaaggcttttatccgagcgatatt gcggtggaatgggaaagcaacggccagccggaaaacaactataaaaccaccccgccggtg ctggatagcgatggcagcttttttctgtatagcaaactgaccgtggataaaagccgctgg cagcagggcaacgtgtttagctgcagcgtgatgcatgaagcgctgcataaccattatacc cagaaaagcctgagcctgagcccgggcaaa (SEQ ID NO: 37).

[0089] In some embodiments, the isolated nucleic acid disclosed herein encodes one or more of the amino acid sequences of any one of SEQ ID NOs: 29 - 34. In some embodiments, the isolated nucleic acid comprises a sequence with at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 38. In some embodiments, the isolated nucleic acid comprises SEQ ID NO: 38.Exemplary BPI-Fc polynucleotide sequence gtgaacccgggcgtggtggtgcgcattagccagaaaggcctggattatgcgagccagcag ggcaccgcggcgctgcagaaagaactgaaacgcattaaaattccggattatagcgatagc tttaaaattaaacatctgggcaaaggccattatagcttttatagcatggatattcgcgaa tttcagctgccgagcagccagattagcatggtgccgaacgtgggcctgaaatttagcatt agcaacgcgaacattaaaattagcggcaaatggaaagcgcagaaacgctttctgaaaatg agcggcaactttgatctgagcattgaaggcatgagcattagcgcggatctgaaactgggc agcaacccgaccagcggcaaaccgaccattaccgcgagcagctgcagcagccatattaac agcgtgcatgtgcatattagcaaaagcaaagtgggctggctgattcagctgtttcataaa aaaattgaaagcgcgctgcgcaacaaaatgaacagccaggtgtgcgaaaaagtgaccaac agcgtgagcagcgaactgcagccgtattttcagaccctgggcggcggcagcgcgagcacc aaaggcccgagcgtgtttccgctggcgccgagcagcaaaagcaccagcggcggcaccgcg gcgctgggctgcctggtgaaagattattttccggaaccggtgaccgtgagctggaacagc ggcgcgctgaccagcggcgtgcatacctttccggcggtgctgcagagcagcggcctgtat agcctgagcagcgtggtgaccgtgccgagcagcagcctgggcacccagacctatatttgc aacgtgaaccataaaccgagcaacaccaaagtggataaaaaagtggaaccgaaaagctgcgataaaacccatacctgcccgccgtgcccggcgccggaagcggcgggcggcccgagcgtg tttctgtttccgccgaaaccgaaagataccctgatgattagccgcaccccggaagtgacc tgcgtggtggtggatgtgagccatgaagatccggaagtgaaatttaactggtatgtggat ggcgtggaagtgcataacgcgaaaaccaaaccgcgcgaagaacagtataacagcacctat cgcgtggtgagcgtgctgaccgtgctgcatcaggattggctgaacggcaaagaatataaa tgcaaagtgagcaacaaagcgctgccggcgccgattgaaaaaaccattagcaaagcgaaa ggccagccgcgcgaaccgcaggtgtataccctgccgccgagccgcgatgaactgaccaaa aaccaggtgagcctgacctgcctggtgaaaggcttttatccgagcgatattgcggtggaa tgggaaagcaacggccagccggaaaacaactataaaaccaccccgccggtgctggatagc gatggcagcttttttctgtatagcaaactgaccgtggataaaagccgctggcagcagggc aacgtgtttagctgcagcgtgatgcatgaagcgctgcataaccattatacccagaaaagc ctgagcctgagcccgggcaaa (SEQ ID NO: 38).

[0090] In some embodiments, the isolated nucleic acid disclosed herein encodes one or more of the amino acid sequences of any one of SEQ ID NOs: 12 - 16. In some embodiments, the isolated nucleic acid disclosed herein encodes one or more of the amino acid sequences of any one of SEQ ID NOs: 19 - 34.

[0091] In some embodiments, the isolated nucleic acid comprises a heterologous promoter. Nonlimiting examples of heterologous promoters include cytomegalovirus (CMV), CMV- immediately early (CMV-IE), CMV / EFl-a, and simian virus 40 (SV40) promoters. In some embodiments, the heterologous promoter is a CMV promoter.

[0092] The isolated nucleic acids described herein are prepared using standard techniques well known to those of skill in the art. The recombinant polypeptide sequences may be used to determine appropriate nucleic acid sequences encoding the particular recombinant polypeptide disclosed thereby. The nucleic acid sequence may be optimized to reflect particular codon preferences for various expression systems according to standard methods well known to those of skill in the art.Vectors

[0093] The present disclosure also provides vectors comprising one or more isolated nucleic acid molecules described herein. The vector can be any non-viral or viral vector known in the art or described herein.

[0094] Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.

[0095] In some embodiments, the vector is suitable for directing the production of a protein product in a cell. In some embodiments, the vector is a non-viral vector. Non-limiting examples of non-viral vectors include a transposon vector, naked DNA (e.g., a DNA plasmid), a liposome, or a lipid nanoparticle.

[0096] In some embodiments, the vector is a viral vector. In some embodiments the viral vector is an adeno-associated virus vector (AAV), an adenoviral vector (AV), a lentiviral vector (LV), a retroviral vector (RV), a herpes simplex virus vector (HSV), or a poxvirus vector.

[0097] For example, viral and non-viral vectors and delivery systems are described in Sung & Kim 2019, Biomaterials Research 23:8; Mali, 2013, Indian Journal of Human Genetics, 19(1):3- 8; Hardee et al., 2017, Genes 8:65; Bulcha et al., 2020, Signal Transduction and Targeted Therapy; Ghosh et al., 2020, Applied Biosafety: Journal of ABSA International 25(1):7- 18, the disclosures of each of which are hereby incorporated by reference herein in their entireties.

[0098] In some embodiments, the vector is a recombinant vector.

[0099] In some embodiments, the vector comprises a transposon system. Non-limiting examples of transposon systems include PiggyBac, Sleeping Beauty, Frog Prince, Himarl, Passport, Minos, hAT, Toll , Tol2, AciDs, PIF, Harbinger, Harbinger3-DR, and Hsmarl , and any of their respective derivatives with equal, lower and / or higher transposition activity. In some embodiments, the transposon system comprises two elements: a DNA element flanked by two terminal inverted repeats, and a transposase that catalyzes the transposon’s mobilization.

[0100] In some embodiments, the transposon system is a PiggyBac transposon system. The PiggyBac transposon system may be isolated from cabbage looper moth Trichoplusia ni, or may be derived from said transposon system. Non-limiting examples of a PiggyBac transposon system are described in (Yusa. Microbiol Spectr. 2015. 3(2):MDNA3-0028-2014 and / or Matteo et al. Methods Mol Biol. 2012. 859:241-54) The PiggyBac transposon system may comprise, for example, a wild-type or a mammalian codon-optimized version of the transposase.

[0101] In some embodiments, the vector comprises an inducible expression system. Non-limiting examples of inducible expression systems include cumate, tetracycline, rapamycin, FKCsA, abscisic acid, tamoxifen, blue light, and riboswitch-inducible expression systems. In some embodiments, the inducible expression system comprises a cumate switch combined with theEFla-CymR repressor-T2A-Puro cassette. The cumate switch system provides tight control through the binding of the CmR repressor in the absence of cumate.

[0102] In some embodiments, the vector comprises a target-specific nuclease. Non-limiting examples of target-specific nucleases include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, and clustered regularly interspaced short palindromic repeats (CRISPR) associated (Cas) nucleases.

[0103] In some embodiments, the vector comprises a CRISPR nuclease and "single guide RNA" or "sgRNA" that guides the Cas nuclease to a desired nucleic acid sequence. In some embodiments, the Cas nuclease has DNA cleavage activity.

[0104] Non-limiting examples of Cas nucleases include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, , Cpfl, C2c3, C2c2 and C2clCsyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Cpfl, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologs thereof, variants thereof, mutants thereof, and derivatives thereof. There are three main types of Cas nucleases (type I, type II, and type III), and 10 subtypes including 5 type I, 3 type II, and 2 type III proteins (see, e.g., Hochstrasser and Doudna, Trends Biochem Sci, 2015:40(l):58-66). Type II Cas nucleases include, but are not limited to, Casl, Cas2, Csn2, and Cas9. These Cas nucleases are known to those skilled in the art. For example, the amino acid sequence of the Streptococcus pyogenes wild-type Cas9 polypeptide is set forth, e.g., in NBCI Ref. Seq. No. NP 269215, and the amino acid sequence of Streptococcus thermophilus wild-type Cas9 polypeptide is set forth, e.g., in NBCI Ref. Seq. No. WP_011681470.

[0105] In some embodiments, the vector comprises one or more promoters, one or more poly-A sequences, one or more selectable markers, and / or an origin. Selection for cells comprising the vector is facilitated by incorporation into the vector of a gene whose product allows the transfected cells to survive and grow under selective conditions. A number of such genes have been identified. These include, among others: (1) neo, a prokaryotic gene which encodes resistance to the aminoglycoside antibiotic G418; (2) E. coli guanine phoshporibosyl transferase (gpf), which encodes resistance to mycophenolic acid (MPA) in the presence of xanthine, [Mulligan et al , Proc. Nat. Acad. Sci. USA, 7 .2Q'12-2 16 (1981)]; (3) dihydrofolate reductase (DHFK), which allows for growth of DHFR" cells in the absence of nucleosides and gene amplification in the presence of increasing concentration of methotrexate; (4) the hisD gene of Salmonella typhimurium which allows growth in the presence of histidinol (Hartman et al. , Proc. Nat. Acad. Sci. USA, 85:8047-8051, (1988)); (5) the trpB gene of E. coli (Hartman et al., Proc. Nat. Acad.Sci. USA, 85:8047-8051, (1988)), which allows growth in the presence of indole (without tryptophan); (6) the glutamine synthetase gene, which allows growth in media lacking glutamine, (7) a puromycin resistance gene (e.g., N-acetyltransferase pac]), and 8) an ampicillin resistance gene (e.g., bld). The availability of these selective markers, either alone or in various combinations, provides flexibility in the generation of mammalian cell lines which express recombinant products at high levels.Cells

[0106] The present disclosure also provides cells comprising one or more isolated nucleic acid molecules described herein. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell (e.g., a murine, bovine, simian, porcine, equine, ovine, or human cell). In some embodiments, the cell is a human cell. In some embodiments, the cell is suited for the manufacture of therapeutic proteins, such as Chinese hamster ovary (CHO) cells, HEK cells, or mouse myeloma cells. In some embodiments, the cell is a CHO cell.

[0107] Polynucleotides and / or vectors described herein can be introduced into a cell by any method known in the art. Non-limiting examples of such methods are transfection, viral infection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro-injection, nanoparticle-mediated nucleic acid delivery, and the like.

[0108] In some embodiments, the polynucleotides and / or vectors direct the production of a protein product in the cell. In some embodiments, the polynucleotides and / or vectors are introduced into a cell by transfection. The transfection protocol may be any known in the art (e.g., those described in Kim and Eberwine. AnalBional Chem. 2010; 397(8):3173-3178).

[0109] In some embodiments, the polynucleotides and / or vectors are introduced using a method that comprises integration of the polynucleotide or a portion thereof into the genome of the cell. In some embodiments, the polynucleotide or a portion thereof is stably integrated, (e.g., covalently inserted into a chromosome by homologous recombination, non-homologous end joining, transposition, etc).

[0110] After introduction to a cell of one or more polynucleotides and / or vectors described herein, cells are grown under conditions suitable for expression of the BPI polypeptide. Suitable methods are known in the art (see, e.g., W02012035079A1, WO2013186371 Al, and WO2012167192A2).In some embodiments, the cells are grown in suspension. In some embodiments, the cells are grown in serum-free media. In some embodiments the cells are grown in the presence of serum.Methods of makingPEGylation

[0111] PEGylation is the conjugation of polyethylene glycol (PEG) to proteins. A variety of chemistries exist for coupling PEGs of various sizes to proteins. For example, PEGs can be attached to proteins at the N-terminus, the C-terminus, or unpaired cysteines. PEGs can be attached to proteins at lysines and cysteines via NHS-ester or maleimide activated PEGs, respectively. Methods for PEGylating proteins are known in the art and can be applied to the compositions disclosed herein, see for example Roberts, M. J. et al. (2002) “Chemistry for peptide and protein PEGylation” Adv. Drug Deliv. Rev. 54, 459-476, Kinstler, O. et al. (2002) “Mono-N- terminal poly(ethylene glycol)-protein conjugates” Adv. Drug Deliv. Rev. 54, or European patents EP0154316 and EP0401384.Purification

[0112] The recombinant polypeptides and variants described herein may be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. A variety of purification techniques for recombinant polypeptides are known in the art.

[0113] The purification technique may comprise a step to clear the cell lysate. For example, the cell culture may be passed through a filter (e.g., a 0.22 or 0.44 pM filter) to yield a cleared cell lysate. The purification technique may additionally comprise a binding step in which the recombinant polypeptide is contacted with a first surface that interacts with it based on a first property of the recombinant polypeptide (e.g., charge, size, hydrophobicity, or affinity). The purification technique may additionally comprise a wash step, in which a first buffer is applied to the first surface, wherein the first buffer selectively removes polypeptides from the first surface while not disrupting the interaction between the first surface and the recombinant polypeptide. The purification technique may additionally comprise an elution step, in which a second buffer is applied to the first surface that disrupts the interaction between the first surface and the recombinant polypeptide. Optionally, the purification may additionally comprise further steps in which the recombinant polypeptide is contacted with a second surface that interacts with it based on a second property of the recombinant polypeptide, optionally wherein further steps comprise a wash step and elution step.

[0114] Example 2 provides an exemplary embodiment of such a purification technique.Terms and concepts

[0115] A number of terms and concepts are discussed below. They are intended to facilitate the understanding of various embodiments of the present disclosure in conjunction with the rest of the present document and the accompanying figures. These terms and concepts may be further clarified and understood based on the accepted conventions in the fields of the present disclosure, as well as the description provided throughout the present document and / or the accompanying figures. Some other terms can be explicitly or implicitly defined in other sections of this document and in the accompanying figures and may be used and understood based on the accepted conventions in the fields of the present disclosure, the description provided throughout the present document and / or the accompanying figures. The terms not explicitly defined can also be defined and understood based on the accepted conventions in the fields of the present disclosure and interpreted in the context of the present document and / or the accompanying figures.

[0116] The terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise.

[0117] The term “or” as used herein, including the claims, is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0118] The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.

[0119] The terms “about” and “approximately” are used as equivalents. Any numerals used in this application with or without about / approximately are meant to cover any normal fluctuations appreciated by one of ordinary skill in the relevant art. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0120] Numerical ranges are provided for certain quantities in the present application. It is to be understood that these ranges comprise all subranges therein. Thus, the range “10 to 400,” also referred to as “10 - 400,” includes all possible ranges therein (e.g., 11-399, 12-398, 13-397, 14- 396, 15-395, 20-300, 40-200, etc.). Furthermore, all values within a given range may be an endpoint for the range encompassed thereby (e.g., the range 10-400 includes the ranges with endpoints such as 10-300, 50-400, etc.). All ranges provided herein also include all discreet numerical values that fall within the given range, including the endpoints (e.g., the range 10 - 400 includes 10, 50, 60, 120, 150, 180, 240, 300, 400, etc.).

[0121] The term "sequence identity" and the related terms and expressions used in the context of describing nucleic acid or amino acid sequences refer to a sequence that has at least 60% sequence identity to a reference sequence. Examples include at least: 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity, as compared to a reference sequence using the programs for comparison of nucleic acid or amino acid sequences, such as BLAST using standard parameters. For sequence comparison, typically one sequence acts as a reference sequence (subject sequence) to which test sequences (query sequence) are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default (standard) program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Methods of alignment of sequences for comparison are well- known. Optimal alignment of sequences for comparison may be conducted, for example, by the local homology algorithm of Smith and Waterman, 1981, by the homology alignment algorithm of Needleman and Wunsch, 1970, by the search for similarity method of Pearson and Lipman, 1988, by computerized implementations of these algorithms (for example, BLAST), or by manual alignment and visual inspection. Algorithms that are suitable for determining percent sequence identity and sequence similarity include BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1990, and Altschul et al., 1977, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site.

[0122] The terms “Fc” or “Fc fragment” refer to a polypeptide including the constant region of an antibody excluding the first constant region immunoglobulin domain. Fc region generally refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last threeconstant region immunoglobulin domains of IgE and IgM. An Fc region may also include part or all of the flexible hinge N-terminal to these domains. For IgA and IgM, an Fc region may or may not include the tailpiece, and may or may not be bound by the J chain. In some embodiments, the Fc region for IgG includes immunoglobulin domains Cgamma2 and Cgamma3 (Cy2 and Cy3) and the lower part of the hinge between Cgammal (Cyl) and Cy2. In some embodiments, the Fc region for IgA includes immunoglobulin domains Calpha2 and Calpha3 (Ca2 and Ca3) and the lower part of the hinge between Calphal (Cal) and Ca2. In some embodiments, the Fc can be a mutant IgG Fc domain that does not dimerize under physiological conditions.

[0123] As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C-C 10) alkoxy- or aryloxypolyethylene glycol or polyethylene glycol-maleimide.

[0124] The terms “treatment” or “treating” used to refer to treatment or treating of a subject indicate any type of intervention or process performed on, or the administration of an active agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, or slowing down the onset, progression, development, severity or recurrence of a symptom, complication or condition, or biochemical indicia associated with a disease or disorder. In some embodiments, “treatment” or “treating” includes a partial remission. In some embodiments, “treatment” or “treating” includes a complete remission.

[0125] The term "administering," as used herein, refers to introducing a recombinant polypeptide, composition, and / or pharmaceutical composition into a subject.

[0126] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the scope of those of skill in the art. Such techniques are explained fully in the literature, such as, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (M. J. Gait, ed., 1984); "Animal Cell Culture" (R. I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (D. M. Weir & C. C. Blackwell, eds.); "Gene Transfer Vectors for Mammalian Cells" (J. M. Miller & M. P. Calos, eds., 1987); "Current Protocols in Molecular Biology" (F. M. Ausubel et al., eds., 1987); "PCR: The Polymerase Chain Reaction", (Mullis et al., eds., 1994); and "Current Protocols in Immunology" (J. E. Coligan et al., eds., 1991), each of which is expressly incorporated by reference herein.EXAMPLESExample 1 : Transfection of CHO cells for stable expression of BPI and variant constructs

[0127] Synthetic genes encoding bactericidal permeability increasing protein (BPI), BPI-His and BPI-Fc are cloned into the PiggyBac (PB) Transposon Vector system using standard methods. Exemplary amino acid sequences for BPI, BPI-His, and BPI-Fc are provided in Table 1. The PiggyBac Transposon System uses a cut-and-paste mechanism to transfer DNA from the PiggyBac Vector into the genome. The Super PiggyBac Transposase recognizes transposonspecific inverted terminal repeats (ITRs) and efficiently integrates the ITRs and intervening DNA into the genome at TTAA sites. The transposon vector is under the control of a CMV promoter and includes a selectable marker (puromycin). A schematic of an exemplary vector is shown in FIG. 1. An inducible PiggyBac vector which features an ultra-tight cumate switch combined with the EFla-CymR repressor-T2A-Puro cassette is used to establish stable cell lines. Expression of BPI and variant constructs is achieved by adding cumate to the cells.

[0128] Briefly, 50pl of serum-free Dulbecco's Modified Eagle Medium (DMEM), 0.5pg of PB Transposon vector containing the respective genes, 0.2pg of PiggyBac Transposase vector (PB210PA-1), and 8.0pl of PureFection (a lipid-based transfection reagent) are combined and vortexed. The mixture is left to stand at room temperature for a sufficient amount of time, or about 15 minutes, to allow the PureFection / DNA complexes to form. The mixture containing the complexes is then added drop-wise to a culture of CHO cells that is about 80% confluent. The mixture is then gently swirled to disperse it over the cells. The transposon vector is integrated into the genome by the PiggyBac transposase.Example 2: Purification of BPI and variant BPI proteins

[0129] Culturing medium from CHO cell cultures expressing BPI, BPI-His, or BPI-Fc are collected and passed through a 0.22 or 0.45pm filter to yield a cleared cell lysate.

[0130] BPI is purified from the cleared cell lysate as previously described (Horwitz et. al., “Expression and characterization of cysteine-modified variants of an aminoterminal fragment of bactericidal / permeability-increasing protein.” Prot Expr Purif, 1996; 8: 28-40.) with the addition of a Heparin HiTrap affinity column. Briefly, large sterile SP-Sepharose beads are added at 1.5% (V / V) to the CHO cell cultures during fermentation. After the fermentation is terminated, the beads are harvested, allowed to settle, and washed several times with 10 mM Na phosphate / 0.15 M NaCl, pH 7.0. The washed beads are packed into a column, washed with 10 mM Na phosphate, 0.25 M NaCl, pH 7.0, and eluted with the same buffer containing 0.8 M NaCl, 5 mM glycine. The eluate is then loaded onto a CM-spherodex column and washed with (1) 10 mM Na phosphate,0.25 M NaCl, pH 7.0; (2) 20 mM Na acetate, 0.2 M NaCl, pH 4.0; and (3) 20 mM Na acetate, 0.3 M NaCl, pH 4.0 in the respective order. The sample is eluted with 1.0 M NaCl in 20 mM Na acetate, 0.3 M NaCl, pH 4.0, concentrated using a Centricon membrane with a 10,000 MW cutoff, and loaded onto a Sephacryl S-100 column. The Sephacryl S-100 column is equilibrated with 5 mM Na citrate, 0.15 MNaCl, pH 5.0. and the sample eluted. The eluate from the Sephacryl S-100 column is then further purified by affinity chromatography using a HiTrap Heparin HP column.

[0131] BPI-His is purified by affinity chromatography using a Ni-NTA Spin column. Briefly, a Ni-NTA Spin column is equilibrated. The cleared cell lysate is loaded onto the column allowing the His-tagged BPI to bind and other proteins to pass through the matrix. The column is then washed and the His-tagged BPI proteins are eluted with an eluting buffer under native or denaturing conditions to yield greater than 95% pure protein. Buffers used to purify BPI-His are listed in Table 2.Table 2: Buffers for purification of BPI-His

[0132] BPI-Fc is purified by affinity chromatography on a Protein-A column using standard protocols. Briefly, the column is flushed with an equilibration buffer / wash buffer, such as a buffer containing 10-50mM phosphate or Tris with a pH between 6.0 to 9.0. To prevent nonspecific adsorption, 0.1 - 0.2M NaCl or KC1 can be added to the buffer. The cleared cell lysate from CHO cells expressing BPI-Fc is loaded onto the pre-washed column. The column is then washed and the BPI-Fc proteins are eluted with an elution buffer. An elution buffer contains between 25-100 mM phosphate, at a pH of between 2.0-3.5, with or without sodium chloride up to 150 mM. Otherelution buffer components that may be used include hydrochloric acid, glycine, citrate, acetate, or other components that buffer well at low pH.Example 3: PEGylation of His-tagged BPI

[0133] BPI-His is PEGylated at the histidine tags as previously described in Cong, et al., "Sitespecific PEGylation at histidine tags". Bioconjugate Chemistry, 23(2):248-63 (2012). Briefly, PEG-bis-sulfone undergoes site-specific conjugation to the histidines in the His-tag through bis- alkyation with the nitrogens on the imidazole ring of histidine to create a covalent conjugate. BPI- His-PEG is then separated from non-conjugated BPI-His by size exclusion chromatography.Example 4: In vivo testing of recombinant BPI proteins

[0134] In vivo mouse models were used to determine the ability of recombinant BPI proteins to promote hematopoietic recovery after exposure to radiation and in GvHD. Recombinant BPI, recombinant BPI-His, and recombinant BPI-Fc are constituted into a formulation buffer. To prepare the formulation buffer, 0.33g / L citric acid, l.Olg / L sodium citrate, 8.76g / L sodium chloride, 2.0g / L Pol oxamer Pl 88, and 2.0g / L polysorbate 80 are dissolved in water. The buffer is adjusted to a pH of about 5.0 and filter sterilized.Radiation induced Myeloablation with thrombocytopenia and neutropenia

[0135] RT-1042, a recombinant BPI-Fc-fusion molecule, was tested for its effects on the survival in mice after Total Body Irradiation (TBI). A schematic showing the structure of RT-1042 is shown in FIG. 2 and the amino acid sequence of RT-1042 is provided as SEQ ID NO: 34. Female BALB / C mice received 6Gy of irradiation. RT-1042 or vehicle control was administered subcutaneously (Sub-Q or sc) within 24 hrs of TBI and continued daily for 10 days. Formulation buffer was used as vehicle control unless otherwise indicated. As shown in FIG. 3, untreated animals did not live past day 16, and only one out of six vehicle-treated mice was alive whereas all animal treated with RT-1042 were alive 30 days after receiving the first Sub-Q administration of RT-1042.

[0136] Female BALB / C mice that received 6Gy of irradiation were left untreated, treated with vehicle control, or treated daily for 10 days with RT-1042 or RT-127, a his-tagged BPI (His-BPI). The amino acid sequence of RT-127 is provided as SEQ ID NO: 44 To study their effects on hematopoietic recovery after exposure to radiation, peripheral blood was collected on day 30 and blood cell counts and hemoglobin levels were determined by an automated hematology analyzer. As shown in Table 3 below, white blood cells (WBC), neutrophils, monocytes, and platelets counts and hemoglobin levels observed in the peripheral blood of mice treated with RT-1042 orRT-127 were comparable to that of mice that did not receive irradiation. RT-1042 and RT 127 restored peripheral blood counts to normal levels by day 30 following 10 days of treatment with RT-1042 or RT-127, following 6 Gy TBI.Table 3: Effect of RT-1042 and RT-127 on Peripheral Blood Counts Following 6 Gy TBIStem cell transplantation and Graft versus Host Disease (GvHD)

[0137] Prior to stem cell transplantation, patients often receive TBI as a conditioning regimen. As illustrated in FIG. 4, Total Body Irradiation (TBI) results in the destruction of the intestinal epithelia and the release of endotoxin from Gram Negative bacteria, triggering a proinflammatory cascade. Without being held to theory, recombinant BPI molecules can neutralize endotoxin and cause the release of IL-22 from dendritic cells.

[0138] As a model of autologous stem cell transplantation, 5 million BALB / C splenocytes and 10 million BALB / C bone marrow cells (BM cells) were administered as 200 pL intravenous injections to BALB / C mice following a radiation dose of 5Gy, administered in 2 split doses of 2.5Gy each.

[0139] As a model of allogenic stem cell transplantation, 5 million BALB / C splenocytes and 10 million BALB / C bone marrow cells were administered as 200 pL intravenous injections to C57BL / 6 mice following a radiation dose of 5Gy, administered in 2 split doses (“bid”) of 2.5Gy each. Mice were then treated with different BPI molecules, including RT-1042 (a BPI-Fc fusion molecule), RT-1274 (BPI-PEG), and RT-127 (BPI-His). RT-1274 is comprised of a recombinant polypeptide attached to PEG and the amino acid sequence of said recombinant polypeptide is provided as SEQ ID NO: 43. Table 4 below provides the details of the experiments.

[0140] Briefly, mice underwent total body irradiation topically, autologous or allogenic cells were injected into the mice intravenously (iv) the day after radiation, and various BPI molecules were administered subcutaneously (sc) twice a day (“bid”) for up to 10 days. Each dose administeredwas at 2.5mg / kg, with the exception of the second dose of the day for Group 4, which was administered at 1.6mg / kg.Table 4: Experimental details for mouse model of allogenic stem cell transplantation and GvHDAllogenic Graft versus Host Disease

[0141] The MHC mismatched mouse model of T cell-mediated acute GVHD (aGVHD) described above where C57BL / 6 host mice are lethally irradiated and engrafted with MHC mismatched T cells from BALB / c donor mice was used to assess the effects of recombinant BPI on aGVHD. At the end of the treatment period, the mice were assessed by the following: (a) body weight, and (b) Kaplan-Meier survival analysis. Further analyses include disease scoring and histopathology. As shown in Table 5 below and in the Kaplan-Meier plot of FIG. 5, treatment with RT-1042 resulted in a 10.7% body weight (BW Nadir) loss and 0 mortality after 35 days (n Remaining on Day 35), treatment with RT-1274 resulted in a 23% body weight loss and 84% mortality after 35 days, and treatment with RT-127 resulted in a 12.7 % body weight loss and 33% mortality after 35 days. Untreated BALB / C allogenic transplanted mice showed a 25.5% loss in body weight and 66% mortality after 35 days.Table 5: Effects of innate immune modulators in a mouse model of allogenic stem cell transplantation and GvHD

[0142] ILS = Increase in Life Span; TTE = Time to Event; BW = Body Weight; TR = Treatment- Related; NTR = Non-Treatment-RelatedExample 5: Neutralization of endotoxin by BPI

[0143] To determine the ability of recombinant BPI to neutralize endotoxin, 5 EU of LPS was incubated with various concentrations of RT-1042 or RT-127. As shown in FIG. 6, 1 nM of both RT-1042 (closed circles) and RT-127 (open circles) resulted in complete neutralization of LPS as measured by the PyroGene™ Recombinant Factor C Endpoint Fluorescent Assay (PyroGene rFC assay) from Lonza.Example 6: Additional in vivo testing of recombinant BPI proteins

[0144] Various in vivo mouse models are used to determine the ability of the recombinant BPI proteins to promote hematopoietic recovery after exposure to radiation and / or chemotherapy and in immune disorders such as GvHD, immune thrombocytopenia, Myelodysplastic Syndromes, Fanconi Anemia, acute radiation syndrome, gastrointestinal-acute radiation syndrome, drug resistant gram-negative infections, and sepsis. To prepare the formulation buffer, 0.33g / L citric acid, l.Olg / L sodium citrate, 8.76g / L sodium chloride, 2.0g / L Poloxamer P188, and 2.0g / L polysorbate 80 are dissolved in water. The buffer is adjusted to a pH of about 5.0 and filter sterilized. Recombinant BPI, recombinant BPI-His, and recombinant BPLFc are constituted into the formulation buffer.Chemotherapy induced thrombocytopenia and neutropenia

[0145] Male BALB / c mice are acclimated prior to chemotherapy at age 12 weeks. Mice are administered 3 doses of cisplatin on alternating days (for example, one dose each on Monday, Wednesday, and Friday). Each dose is 5mg / kg, given intraperitoneally. After about 24 hours, mice are either treated with vehicle control (formulation buffer) or received one or more of thefollowing treatments for 10 days: 1) recombinant BPI, 2) recombinant BPI-His, or 3) recombinant BPI-Fc. Treatments are administered twice daily, six to eight hours apart, through subcutaneous injection. Each mouse is administered 250 pl of a 2 mg / ml stock constituted in formulation buffer per injection. All mice are observed at least twice daily. Blood is collected from each mouse at the end of the treatment period and neutrophil and platelet count are determined by an automated hematology analyzer.Fanconi Anemia

[0146] A mouse model of Fanconi anemia is used to test the efficacy of BPI analogs in restoring normal platelet counts. ARGN mice lack exon 37 of the Fanconi anemia, complementation group A (FANCA) gene and are maintained on a NOD.Cg-RagltmlMomI12rgtmlwj1 / SzJ background. FANCA is a member of the Fanconi anemia (FA) complementation group (FANC) which makes up a multi-protein nuclear complex involved in cellular responses to DNA damage and germ cell survival. Mutations in this gene are the most common cause of Fanconi anemia, a heterogeneous recessive disorder characterized by cytogenetic instability, hypersensitivity to DNA crosslinking agents, increased chromosomal breakage, and defective DNA repair. This strain is an immune deficient model of Fanconi’s Anemia which may be used as recipients for human xenografts, which has been shown to give recipients a profound selective growth advantage.

[0147] The mice are either treated with vehicle control (formulation buffer) or received one or more of the following treatments for up to 10 days: 1) recombinant BPI, 2) recombinant BPI-His, or 3) recombinant BPI-Fc. Treatments are administered twice daily, six to eight hours apart, through subcutaneous injection. Each mouse is administered 250 pl of a 2 mg / ml stock constituted in formulation buffer per injection. Blood is collected from each mouse at the end of the treatment period and platelet count is determined by an automated hematology analyzer.Drug Resistant Gram-Negative Infection

[0148] A murine model of thigh infection is used to test the antimicrobial efficacy of BPI analogs. This model is validated with clinically relevant strains, including E. coli. Klebsiella and S. aureus (both MRSA and MSSA). All currently used antimicrobial classes have been evaluated in this model.

[0149] Mice are rendered neutropenic by cyclophosphamide treatment prior to infection and are infected with drug resistant S. aureus via intramuscular injection. Within 24 hours of infection, mice are treated with vehicle control (formulation buffer) or received one or more of the following treatments for 10 days: l) recombinant BPI, 2) recombinant BPI-His, or 3) recombinant BPI-Fc. Treatments are administered twice daily, six to eight hours apart, through subcutaneous injection.Each mouse is administered 250 pl of a 2 mg / ml stock constituted in formulation buffer per injection. At the end of the treatment period, the mice are assessed by one or more of the following: (a) body weight, (b) clinical scores for impaired mobility, (c) bacterial load (CFU) in the thigh muscle, and (d) gross pathology.

[0150] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0151] All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.

[0152] From the foregoing, it will be appreciated that specific embodiments of the invention have been described herein for purposes of illustration, but that various modifications may be made without deviating from the scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

CLAIMSI / We claim:

1. A recombinant polypeptide, comprising:(a) an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1-5; and(b) an Fc fragment.

2. The recombinant polypeptide of claim 1, wherein the amino acid sequence comprises any one of SEQ ID NOs: 1 - 5.

3. The recombinant polypeptide of claim 1 or claim 2, wherein the Fc fragment is human IgG.

4. The recombinant polypeptide of claim 3, wherein the Fc fragment is human IgGl .

5. The recombinant polypeptide of any one of claims 1 - 4, wherein the Fc fragment comprises an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 6.

6. The recombinant polypeptide of claim 5, wherein the Fc fragment comprises an amino acid sequence comprising SEQ ID NO: 6.

7. The recombinant polypeptide of any one of claims 1- 6, wherein the recombinant polypeptide is linked to the Fc fragment by a linker.

8. The recombinant polypeptide of claim 7, wherein the linker is a Glycine-Serine (Gly- Ser) rich linker.

9. The recombinant polypeptide of claim 8, wherein the linker is a Gly-Ser linker.

10. The recombinant polypeptide of claim 8 or claim 9, wherein the linker comprises a sequence selected from the group consisting of: GSAGSAAGSGEF (SEQ ID NO: 39), (GGGGS)n (SEQ ID NO: 40), GGGS (SEQ ID NO: 41), and GGGGS (SEQ ID NO: 42).

11. The recombinant polypeptide of claim 10, wherein the linker is GGGS (SEQ ID NO: 41).

12. The recombinant polypeptide of any one of claims 8-11, wherein the amino acid sequence comprises a sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 7-11.

13. The recombinant polypeptide of claim 12, wherein the amino acid sequence comprises any one of SEQ ID NOs: 7-11.

14. A recombinant polypeptide, comprising:(a) an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1-5; and(b) a polyhistidine-tag (His-tag).

15. The recombinant polypeptide of claim 14, wherein the amino acid sequence comprises any one of SEQ ID NOs: 1 - 5.

16. The recombinant polypeptide of claim 14 or claim 15, wherein the His-tag is at the C- terminus or the N-terminus of the recombinant polypeptide17. The recombinant polypeptide of claim 16, wherein the His-tag is at the C-terminus of the recombinant polypeptide.

18. The recombinant polypeptide of any one of claims 14-17, wherein the His-tag is an 8X- His-tag.

19. The recombinant polypeptide of any one of claims 14-18, comprising a sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 12-16.

20. The recombinant polypeptide of claim 19, comprising the sequence of any one of SEQ ID NOs: 12 -16.

21. The recombinant polypeptide of any one of claims 1-20, wherein the amino acid sequence further comprises a sequence encoding a signal peptide.

22. The recombinant polypeptide of claim 21, wherein the sequence encoding the signal peptide comprises SEQ ID NO: 17 or SEQ ID NO: 18.

23. The recombinant polypeptide of claim 21 or claim 22, comprising the sequence of any one of SEQ ID NOs: 19 - 34 or 43-44.

24. The recombinant polypeptide of any one of claims 1-23, wherein the recombinant polypeptide partially or fully inactivates endotoxin.

25. A composition comprising the recombinant polypeptide of any one of claims 1-24.

26. The composition of claim 25, wherein the recombinant polypeptide is covalently attached to polyethylene glycol (PEG).

27. A composition comprising a recombinant polypeptide, wherein the recombinant polypeptide comprises an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1-5; and wherein the recombinant polypeptide is covalently attached to polyethylene glycol (PEG).

28. The composition of claim 27, wherein the recombinant polypeptide comprises any one of SEQ ID NOs: 1-5.

29. The composition of any one of claims 25 - 28, wherein the recombinant polypeptide comprises a His-tag and the PEG is covalently attached to the His-tag.

30. The composition of any one of claims 25 - 29, wherein the PEG is PEG-40.

31. A pharmaceutical composition comprising the recombinant polypeptide of any one of claims 1 - 23, or the composition of any one of claims 25 - 30, and a pharmaceutically acceptable excipient.

32. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the recombinant polypeptide of any one of claims 1 - 24.

33. The isolated nucleic acid molecule of claim 32, comprising one or more of a sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 35 - 38.

34. The isolated nucleic acid molecule of claim 33, comprising one or more of SEQ ID NOs: 35 - 38.

35. The isolated nucleic acid molecule of any one of claims 32 - 34, comprising a heterologous promoter.

36. The isolated nucleic acid molecule of claim 35, wherein the heterologous promoter is selected from the group consisting of: CMV, CMV-IE, CMVZEFlalpha, and SV40.

37. The isolated nucleic acid molecule of claim 36, wherein the heterologous promoter is a CMV promoter.

38. A vector comprising the isolated nucleic acid molecule of any one of claims 32 - 37.

39. The vector of claim 38, wherein the vector is a non-viral vector.

40. The vector of claim 39, wherein the non-viral vector is a transposon vector.

41. The vector of claim 40, wherein the transposon vector is a PiggyBac transposon vector.

42. A cell comprising the isolated nucleic acid molecule of any one of claims 32 - 37 or the vector of any one of claims 38 - 41.

43. A kit comprising the isolated nucleic acid molecule of any one of claims 32 - 37 or the vector of any one of claims 38 - 41, and optionally instructions for use.

44. A method of treating a disease or a disorder, or a symptom thereof, in a subject in need thereof, comprising administering to the subject:(a) the recombinant polypeptide of any one of claims 1 - 24;(b) the composition of any one of claims 25 - 30;(c) the pharmaceutical composition of claim 31;(d) the isolated nucleic acid molecule of any one of claims 32 - 37; or(e) the vector of any one of claims 38 - 41.

45. The method of claim 44, wherein the disease or disorder is selected from the group consisting of: extensively drug-resistant gram-negative infections, graft-vs-host disease (GvHD), Fanconi amenia, chemotherapy induced neutropenia, chemotherapy induced thrombocytopenia, myeloablative radiation therapy induced neutropenia, and myeloablative radiation therapy induced thrombocytopenia.

46. A method of promoting hematopoietic recovery in a subject in need thereof, comprising administering to the subject:(a) the recombinant polypeptide of any one of claims 1 - 24;(b) the composition of any one of claims 25 - 30;(c) the pharmaceutical composition of claim 31;(d) the isolated nucleic acid molecule of any one of claims 32 - 37; or(e) the vector of any one of claims 38 - 41.

47. The method of claim 46, wherein the subject has graft-versus-host disease (GvHD), immune thrombocytopenia, myelodysplastic syndrome (MDS), Fanconi anemia, or acute radiation syndrome (ARS).

48. A recombinant polypeptide, comprising:(a) an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1-5; and(b) an Fc fragment; wherein the recombinant polypeptide partially or fully inactivates endotoxin.

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