WO2019113258A1 - Modified serpin rcl peptides and uses thereof - Google Patents

Abstract

In some aspects, the disclosure relates to compositions and methods that are useful for treating inflammatory disorders and conditions. In some embodiments, the disclosure provides isolated reactive center loop (RCL) peptides derived from Myxoma virus serine proteinase inhibitor (Serp) proteins and methods for treating inflammatory disorders using the same.

Description

MODIFIED SERPIN RCL PEPTIDES AND USES THEREOF

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C 119(e) of the filing date of U.S. Provisional Application Serial No. 62/595,066, filed December 5, 2018, entitled“MODIFIED SERPIN RCL PEPTIDES AND USES THEREOF”, the entire contents of which are incorporated herein by reference.

BACKGROUND

Myxomavirus Serp-l is a secreted, heavily glycosylated 55 kDa protein that contains 369 amino acid residues and belongs to the serpin superfamily. As such, Serp-l shares -30% sequence homology with other serpins, indicating a similar mode of function. The general mechanism of serpin function occurs through a suicide inhibitory interaction between residues of the Reactive Center Loop (RCL; residues 305-331) and targeted proteases. Through the RCL, Serp-l can target thrombolytic proteinases, tissue- and urokinase-type plasminogen activators (tPA and uPA, respectively), plasmin, factor Xa, and thrombin in regulation of coagulation. Serp-l shares some protease targets with the human serpin plasminogen activator inhibitor- 1 (PAI-l, SERPINE1), which is the dominant mammalian inhibitor of tPA and uPA and can also inhibit activated protein C and thrombin in the presence of the glycosaminoglycan heparin sulfate, in addition to targets of anti-thrombin (ATIII, SERPINC1).

Peptides derived from the reactive center loop (RCL) of the Myxoma virus-derived protein Serp-l have been developed as stand-alone therapeutics for reducing vasculitis and improving survival in MHV68-infected mice. However, both Serp-l and the RCL peptides lose activity in MHV68 -infected mice after antibiotic suppression of intestinal microbiota.

SUMMARY

Aspects of the disclosure relate to compositions and methods that are useful for inhibiting serine proteinases, for example human serpins. The disclosure is based, in part, on isolated polypeptides that are derived from, or variants of, Myxomavirus serine proteinase inhibitor 1 (Serp-l). In some embodiments, isolated polypeptides described by the disclosure inhibit certain human serine proteinase enzymes ( e.g ., PAI-l, ATIII, A1AT etc.) and are therefore useful, in some embodiments, for certain treating inflammatory diseases such as vasculitis, lupus, etc.

Accordingly, in some aspects the disclosure relates to an isolated polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of

Myxomavirus Serine Proteinase Inhibitor 1 (Serp-l) as provided in SEQ ID NO: 1, wherein the amino acid sequence of the recombinant protein comprises one or more mutations at position L316, 1317, or L3l6 and 1317.

In some embodiments, an isolated polypeptide is at least 95% or at least 99% identical to the amino acid sequence as set forth in SEQ ID NO: 1.

In some embodiments, one or more mutations are L316D, I317E, or L316D and I317E.

In some embodiments, an isolated polypeptide comprises the sequence set forth in SEQ ID NO:

2 or 3.

In some embodiments, an isolated polypeptide comprises an amino acid sequence having at least 85% identity to SEQ ID NO:4 (GTTASSDTAITX1X2PR); wherein if Xi is leucine, then X₂ is not isoleucine; and wherein if X₂ is isoleucine, then Xi is not leucine.

In some embodiments, Xi is any polar amino acid. In some embodiments, X₂ is any polar amino acid. In some embodiments, Xi is any charged amino acid. In some embodiments, X₂ is any charged amino acid.

In some embodiments, X₂ is glutamic acid. In some embodiments an isolated polypeptide comprises the sequence set forth in SEQ ID NO: 5 (GTTASSDTAITXiEPR).

In some embodiments, Xi is aspartic acid. In some embodiments, Xi is aspartic acid and X₂ is glutamic acid. In some embodiments, an isolated polypeptide comprises the sequence set forth in SEQ ID NO: 6 (GTT AS S DT AITDEPR) .

In some embodiments, an isolated polypeptide inhibits activity of one or more serine proteinase. In some embodiments, a serine proteinase is a human serpin.

In some aspects, the disclosure provides an isolated nucleic acid comprising a nucleic acid sequence encoding an isolated polypeptide as described herein.

In some aspects, the disclosure provides a composition comprising an isolated polypeptide as described herein and a pharmaceutically acceptable carrier.

In some aspects, the disclosure provides a method for inhibiting serine proteinase activity in a cell, the method comprising delivering to the cell an isolated polypeptide as described herein, or a composition as described herein. In some embodiments, a cell is a mammalian cell. In some embodiments, a cell is a human cell or a rodent cell ( e.g ., mouse cell).

In some aspects, the disclosure provides a method for treating an inflammatory disorder or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of an isolated polypeptide as described herein, or a composition as described herein.

In some embodiments, an inflammatory disorder or condition is vasculitis, for example vasculitis characterized by lung hemorrhage, lupus, viral sepsis, or transplant rejection.

In some embodiments, a subject is a mammal. In some embodiments, a subject is a human or a rodent (e.g., a mouse). In some embodiments, a subject has been previously treated with an antibiotic agent. In some embodiments, a subject is characterized by a suppression of gut bacteria.

In some embodiments, one or more isolated polypeptides as described by the disclosure are administered directly to a subject (e.g., administered by injection, such as intravenous injection, intraperitoneal injection, intravascular injection, etc.). In some embodiments, one or more isolated polypeptide as described by the disclosure are administered to a subject using a gene therapy vector (e.g., a gene therapy vector comprising one or more nucleic acids encoding one or more isolated polypeptide as described by the disclosure), for example a viral vector (e.g., an AAV vector or an rAAV) encoding one or more isolated polypeptides as described herein.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows representative structures of RCF-derived peptides and modified S-7 peptides (depicted from N- to C-termini, left to right). Included are S-l (I₃₁₇PRNAF₃₂₂; SEQ ID NO: 7), S-3 (R₃₁₉NAF₃₂₂; SEQ ID NO: 8), S-5 (T ₃₂₃ AIV ANKPF₃₃₁ ; SEQ ID NO: 9), S-7

(G₃₀₅TT AS S DT AITLIPR₃₁₉ ; SEQ ID NO: 10), MPS7-8 (G₃₀₅TT AS S DT AITLEPR₃₁₉ ; SEQ ID NO: 2), and MPS7-9 (G₃₀₅TTASSDT AITDEPR₃₁₉ ; SEQ ID NO: 3).

FIG. 2 shows an overlay of the cleaved Serp-l structure with A) the cleaved structure of PAI-l (PDB: 1A7C; r.m.s.d. = 0.997 A), B) the cleaved structure ATIII (PDB: 2B4X; r.m.s.d. = 1.718 A), and C) the cleaved structure of A1AT (PDB: 4PYW; r.m.s.d. = 1.829 A). Each structure is highly homologous despite differences in amino acid sequences, with the highest level of conservation observed between the five core b-strands. FIG. 3 shows a structure of PAI-l (SEQ ID NO: 11) overlaid with coordinates from S-7 (SEQ ID NO: 10) with its sequence shown. Some residues not visible due to cleavage and excluded from labeling. This includes L316, E317, P318, R319. A) Overlay of residues from Serp-l (SEQ ID NO: 12) and PAI-l that correspond to the region where the S-7 peptide was derived. Highlighted, are residues that are not conserved between Serp-l and PAI-l, including 307, 311, 314, and 315 (Serp-l numbering). B) Main-chain interactions of the residues of Serp- 1 from (A) with the main-chain of adjacent b-strands from PAI-l. H-bonding interactions are shown with the neighboring b-strands from PAI-l.

FIG. 4 shows structural basis of S-l, S-3, and S-5 design and function from the serpin RCL. Highlighted are regions of the RCL corresponding to designed peptides (S-l, S-3, and S- 5) in the A) native (PDB: 5BRR), B) latent (PDB: 1LJ5), and C) cleaved (where the RCL has been inserted as a b-sheet; PDB: 1A7C) forms of PAI-l. The predicted binding sites of RCL peptides S-l and S-3 with protease targets D) uPA and E) tPA are shown. An overlay of residues from PAI-l (SEQ ID NO: 11) and Serp-l (SEQ ID NO: 12) corresponding to positions in S-l and S-3 are shown in (D) and (E) and highlight the variability between each sequence.

FIG. 5 shows the RCL-peptide binding region in target serpins utilized to design MPS peptides (shown as spheres). PAI-l (shown in as a ribbon diagram) was used as a template, due to its high homology with other target serpins, (PDB: 1A7C) exploited via peptide residue modification. Highlighted in left-hand panels are residues corresponding to A) S-7 B) MPS7-8 and C) MPS7-9 docked into the region utilized for MPS design. Presented are an overlays of residues corresponding to serpins PAI-l, ATIII, and A1AT with relative predicted bond distances (in A) shown. This includes distances from position 317 and the conserved Thr 161, 211, and 180 in PAI-l, ATIII, and A1AT, respectively (>4 A in S-7, and <3.5 A in MPS7-8 and MPS7-9), and Asp 158 in PAI-l and Asn 208 in ATIII (>3 A in S-7, and <3 A in MPS7-8 and MPS7-9). In addition, C) shows the relative predicted distances (<3 A) of position 316 in MPS7-9 with residues Arg 162 and Leu 163 in PAI-l, Val 214 in ATIII, and Val 311 in A1AT.

FIG. 6 shows interaction of Serp-l RCL-derived peptides and serpin-protease

complexes. (A) Fluorogenic real-time kinetic assays of Thrombin- ATIII interaction with and without S-7, MPS7-8 and MPS7-9. (B) Non-reducing SDS-PAGE of Thrombin- ATIII complex formation in the presence or absence of S-7, MPS7-8 and MPS7-9.

FIG. 7 shows Kaplan-Meier Survival Curves for S-7, S-8, modified S-7 (MPS7-8 and -9) peptides in MHV68 -infected IFNyR KO mouse model with suppression of gut microbiome by antibiotics (Abx). Data indicate that treatment with MPS7-8 and -9 (modified peptides) recovers some function and improves survival in antibiotic treated MHV68 infected IFNyR KO mice.

FIG. 8 shows a representative sequence alignment of Serp-l with human serpins PAI-l, AT, and A1AT. Alignments were performed using CLUSTAL W. SEQ ID NOs: 13-16 are shown, top to bottom.

FIG. 9 shows a crystallographic arrangement of Serp-l AUs in the C2 unit cell. A) Crystal packing of Serp-l in the C2 unit cell with highlighted“gaps” between each AU. B) Crystal packing of Serp-l similar to (A) with added glycosylation (sticks) shown to occupy spaces between each proteomer. C) Position of a molecule of poly-ethylene glycol (PEG) shown between crystallographic symmetry mates and contributing to the crystal packing of Serp-l. D) Electron density of the observed PEG molecule in the Serp-l structure with the 2Fo- Fc map contoured to an r.m.s.d. of 1.4.

FIG. 10 shows structural basis for the poor inhibitory and therapeutic effects of the S-5 peptide with sequences from Serp-l and PAI-l shown. A) Positions of residues from Serp-l and PAI-l corresponding to residues from the S-5 peptide. B) Positions of residues from (A) shown in a view that has been rotated 90°.

FIG. 11 shows initial structure for the FEP calculations for the A) S-7/AT-III complex (based on cleaved ATIII, PDB: 1ATT) and B. S7/PAI-1 complex (based on cleaved PAI-l PDB: 3CVM). SEQ ID NOs: 17-19 shown, left to right.

FIG. 12 shows predicted structures of L inked glycosylation in Serp-l. A) Model of fully glycosylated (sticks) Serp-l (positions Asn 28, 99, and 172). B) Electron density of observed glycans of Asn 28 (2Fo-Fc map contoured to r.m.s.d. of 1.4). C) Schematic showing the overall construct of Serp-l with the RCL and glycosylation sites marked (sequences or surrounding residues shown).

DETAILED DESCRIPTION

Aspects of the disclosure relate to compositions comprising isolated polypeptides that are derived from, or variants of, Myxomavirus serine proteinase inhibitor 1 (Serp-l). In some aspects, the disclosure provides methods of inhibiting serine proteases by administering compositions described herein. Serpins

Serine protease inhibitors, or serpins, are ubiquitous, complex, and highly active regulatory molecules that effectively control multiple coagulation, inflammatory, and

neuroendocrine pathways. The amino acid sequence in the reactive center loop (RCL) of serpins ( e.g ., amino acid residues 305-331 of SEQ ID NO: 1) can act as bait for target serine proteases initiating structural changes in the serpin/protease complex and culminating in cell suicide inhibition (Silverman et ah, (2001) J Biol Chem 276: 33293-33296; Law et al, (2006) Genome Biol 7: 216; and Gooptu and Lomas (2009) Annu Rev Biochem 78: 147-176). Previous studies have shown significant and prolonged anti-inflammatory functions detected with myxomavirus- derived, Serp-l (Lucas et al., (1996) Circulation 94: 2890-2900; Viswanathan et al., (2006) Thromb Haemost 95: 499-510; and Chen et al., (2013) Antimicrob Agents Chemother 57: 4114- 4127), and mammalian serpin, neuroserpin (NSP), after single dose injections of purified proteins in animal models of vascular disease (Munuswamy-Ramanujam et al., (2010) Thromb Haemost 103: 545-555).

Serp-l is a secreted myxomavirus-derived protein that binds and inhibits urokinase- and tissue-type plasminogen activators (uPA and tPA, respectively), plasmin, and factor X (fXa) with demonstrated inhibition of plaque growth and organ scarring in mouse, rat, and rabbit balloon angioplasty induced neointimal plaque growth and in rodent transplant models (Chen et al., (2013) Antimicrob Agents Chemother 57: 4114-4127). Reactive center loop (RCL) peptides derived from Serp-l, for example S-l (I317PRNAL322; SEQ ID NO: 7), S-3 (R319NAL322; SEQ ID NO: 8), S-5 (T 323 AIV ANKPL331 ; SEQ ID NO: 9), and S-7 (G305TT AS SDT AITLIPR319 ; SEQ ID NO: 10). However, it has been observed under certain circumstances that these peptides function as poor inhibitors of uPA and tPA, and weak inhibitors for PAI- 1. Additionally, it has been observed that after antibiotic treatment to suppress intestinal bacteria (the gut microbiome), activity of certain RCL peptides is lost.

In some aspects, the disclosure relates to isolated polypeptides that are derived from, or variants of, Myxomavirus serine proteinase inhibitor 1 (Serp-l) or Serp-l RCL peptides. In some embodiments, isolated peptides described by the disclosure display extended activity (e.g., relative to previously developed RCL peptides, such as S-7) and/or retained activity in a subject when combined with antibiotic treatments.

“RCL peptides” refers to polypeptides derived from a reactive site loop of a serine protease inhibitor, for example Serp-l. RCL peptides may be a fragment of a Serp-l peptide, or a variant of a RCL peptide, for example a variant of S-7 peptide. A“variant” of a peptide generally refers to a peptide having an amino acid sequence that is at least 90% identical to a Serp-l protein (e.g., SEQ ID NO: 1) or at least 80% identical to corresponding portion of a reference peptide (e.g., the peptide from which the variant is derived) and comprises one or more amino acid substitutions, additions, or deletions relative to the reference peptide. For example, a S-7 RCL peptide variant may comprise an amino acid sequence that is at least 80% identical to a S-7 RCL peptide (e.g., SEQ ID NO: 10), and contains at least one amino acid substitution relative to S-7 RCL peptide.

The skilled artisan recognizes that a number of suitable algorithms (e.g., Multiple Sequence Alignment algorithms) may be used to calculate the sequence identity of a serpin RCL peptide variant, for example BLAST, CLUSTAL, Needleman-Wunsch, Smith-Waterman, LALIGN, etc.

In some embodiments, a serpin RCL peptide variant is at least or about 90% identical (e.g., at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to a Serp-l protein (e.g., SEQ ID NO: 1). In some embodiments, a serpin RCL peptide variant is at least or about 80% (e.g., at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to a corresponding portion of an endogenous serpin protein or serpin-derived peptide (e.g., a serpin RCL peptide such as S-7 peptide).

In some embodiments, a serpin RCL peptide variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions relative to a Serp-l protein (e.g., SEQ ID NO: 1). In some embodiments, a serpin RCL peptide variant comprises 1, 2, or 3 amino acid substitutions relative to a corresponding portion of an endogenous serpin protein or serpin-derived peptide (e.g., a serpin RCL peptide such as S-7 peptide). Generally, an amino acid substitution may be a conservative amino acid substitution or a non-conservative amino acid substitution.

Accordingly, in some embodiments, the disclosure provides an isolated polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of Myxomavirus Serine Proteinase Inhibitor 1 (Serp-l) as provided in SEQ ID NO: 1. In some embodiments, the isolated polypeptide is a S-7 RCL peptide variant. In some embodiments, the isolated polypeptide comprises an amino acid substitution at position L316, position 1317, or positions L316 and 1317 relative to SEQ ID NO: 1. In some embodiments, an amino acid substitution at position L316 relative to SEQ ID NO: 1 is selected from L316K, L316R, L316N, L316Q, L316W, L316Y, L316D, and L316E. In some embodiments, an amino acid substitution at position 1317 relative to SEQ ID NO: 1 is selected from I317D and I317E. In some embodiments, the amino acid sequence of the recombinant protein ( e.g ., S-7 RCL peptide variant) comprises one or more mutations at position L316, 1317, or L316 and 1317. In some embodiments, an isolated polypeptide comprises the sequence set forth in SEQ ID NO: 2 or 3.

In some embodiments, an isolated polypeptide comprises an amino acid sequence having at least 85% identity to SEQ ID NO: 4 (GTTASSDTAITX1X2R); wherein if Xi is leucine, then X₂ is not isoleucine; and wherein if X₂ is isoleucine, then Xi is not leucine. In some

embodiments, Xi is any polar amino acid. In some embodiments, X₂ is any polar amino acid.

In some embodiments, Xi is any charged amino acid. In some embodiments, X₂ is any charged amino acid. In some embodiments, Xi is aspartic acid. In some embodiments, X₂ is glutamic acid. In some embodiments, Xi is aspartic acid and X₂ is glutamic acid. In some embodiments an isolated polypeptide comprises the sequence set forth in SEQ ID NO: 5. In some

embodiments, an isolated polypeptide comprises the sequence set forth in SEQ ID NO: 6.

An RCL peptide variant comprise modified amino acids, such as those known to one of ordinary skill in the art. Disclosed peptides can include natural, unnatural, or non-amino acid residues. Synthetic peptides, for example, include those with modified amino acids or other moieties in place of an amino acid. The inclusion of unnatural or non-amino acids can be made to stabilize the peptide, block metabolization, or to create a conformational change in the peptide which would increase its effectiveness. Preferably, the amino acids of the peptides are in the L- orientation, although amino acids or peptides in the D-orientation can also be used, as can be peptides in the reverse orientation.

In some embodiments, isolated polypeptides (e.g., Serp-l variants and/or RCL peptide variants) as described herein inhibit one or more serine proteases. The serine proteases can be thrombolytic or thrombotic. Examples of serine proteases inhibited by isolated polypeptides described herein include but are not limited to uPa, tPa, anti-thrombin III (ATIII), and al- antitrypsin (A1AT). In some embodiments, isolated peptides described herein reduce inflammation. In some embodiments, a“variant” of an RCL peptide retains the functional activity of the reference polypeptide (e.g., ability to bind human serpins).

The length of an isolated polypeptide may vary. In some embodiments, an isolated polypeptide (e.g., a Serp-l variant or a RCL peptide variant) is about, or less than about, 25 amino acids, or 21 amino acids, or 4 amino acid residues in length. In some embodiments, an isolated polypeptide (e.g., a Serp-l variant or a RCL peptide variant) is between 4 and 25 (e.g.,

4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) amino acids in length. In some embodiments, an isolated polypeptide (e.g., a Serp-l variant or a RCL peptide variant) is 13, 14, or 15 amino acids in length.

An isolated polypeptide (e.g., a Serp-l variant or a RCL peptide variant) disclosed herein can be modified at, for example, either the amino terminus, the carboxy terminus, or both. For example, an amino acid sequence encoding an RCL peptide variant may include at least two cysteine residues, one or both of which are, optionally, at the C-terminal or N-terminal of the RCL peptide variant. In some embodiments, an RCL peptide variant can include a naturally occurring serpin-derived sequence having at or near each of the C- and N-termini, a cysteine residue. The serpin-derived sequence can be cyclized by formation of a disulfide bond between these two cysteine residues (or, more generally, between two of the at least two cysteine residues present at the terminal regions). While the peptides (e.g., RCL peptide variants) of the disclosure can be linear or cyclic, cyclic peptides generally have an advantage over linear peptides in that their cyclic structure is more rigid and hence their biological activity can be higher than that of the corresponding linear peptide. Any method for cyclizing peptides can be applied to the an Serp-l variants or RCL peptide variants described herein.

Alternatively, or in addition, an RCL peptide variant can also include a substituent at the amino-terminus or carboxy-terminus. The substituent can be an acyl group or a substituted or unsubstituted amine group (e.g., the substituent at the N-terminus can be an acyl group and the C-terminus can be amidated with a substituted or unsubstituted amine group (e.g., an amino group having one, two, or three substituents, which may be the same or different)). The amine group can be a lower alkyl (e.g., an alkyl having 1-4 carbons). The acyl group can be a lower acyl group (e.g., an acyl group having up to four carbon atoms), especially an acetyl group.

The fragments of an RCL peptide variant can also be modified in order to improve absorption, including for example, an addition of sugar residues to enhance transport across the blood-brain barrier.

In some embodiments, an RCL peptide variant described by the disclosure further comprises (e.g., is linked to or conjugated to) a heterologous polypeptide (e.g., a polypeptide having a sequence that does not appear in Serp-l or an RCL peptide variant). The heterologous polypeptide can be a polypeptide that increases the circulating half-life of the fragment of the RCL peptide variant to which it is attached ( e.g ., fused, as in a fusion protein). In some embodiments, the heterologous polypeptide is albumin (e.g., a human serum albumin or a portion thereof, bovine serum albumin or a portion thereof, etc.).

Any of the RCL peptide variants described herein can be one of a plurality present in multimeric form (e.g., as a dimer, trimer, 4-mer, etc.). The multimeric form can also include one or more types of RCL peptide variants wherein the two or more variants are identical or non identical.

In some embodiments, an RCL peptide variant is formulated with a physiologically acceptable compositions. In some embodiments, an RCL peptide variant is contained within a composition that is not suitable for administration to a living being (e.g., concentrated stocks or frozen or lyophilized compositions). The physiologically acceptable compositions can be pharmaceutical compositions, and methods of treating patients are described further below. The terms "physiologically acceptable" or pharmaceutically acceptable" are used herein to mean any formulation which is safe, and provides the appropriate delivery for the desired route of administration of an effective amount of a composition described herein.

Nucleic Acids

Aspects of the disclosure relate to nucleic acid molecules that encode the RCL peptide variants described herein. A nucleic acid may be DNA, RNA, or a combination thereof.

Nucleic acids may encode expression vectors, for example a nucleic acid sequence encoding an RCL peptide variant that is operably linked to one or more regulatory elements (e.g., Kozak sequence, promoters, enhancers, terminators, splice-signals, etc.), also referred to as an “expression cassette”. A vector may be a plasmid, cosmid, viral vector, etc. In some embodiments, the disclosure provides an adeno-associated virus (rAAV) vector, or rAAV particle, that comprises an isolated nucleic acid encoding a serpin RCL peptide variant as described by the disclosure.

An expression cassette can include 5' and 3' regulatory sequences operably linked to a polynucleotide disclosed herein. "Operably linked" is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a polynucleotide disclosed herein and a regulatory sequence (e.g., a promoter) is a functional link that allows for expression of a polynucleotide disclosed herein. Operably linked elements can be contiguous or non-contiguous. When used to refer to the joining of two protein coding regions, by operably linked is intended that the coding regions are in the same reading frame. An expression cassette may further comprise at least one additional polynucleotide to be co-transformed into the organism. Alternatively, one or more polypeptide(s) can be expressed on one or more expression cassettes. Expression cassettes can be provided with a plurality of restriction sites and/or recombination sites for insertion of the polynucleotide to be under the transcriptional regulation of the regulatory regions.

In some embodiments, an RCL peptide variant as described by the disclosure is produced by chemical synthesis ( e.g ., not produced using DNA recombinant expression). Methods of peptide synthesis are known, for example as described by Stawikowski et al. Curr. Protoc. Protein Sci. 2002 Feb; CHAPTER: Unit-l8.l.

Pharmaceutical Compositions

Aspects of the disclosure relate to pharmaceutical compositions comprising one or more RCL peptide variants as described herein. Pharmaceutical compositions described herein can be administered in any form by any effective route, including but not limited to, oral, parenteral (e.g., intravenous, intradermal, subcutaneous, intraperitoneal, intramuscular, inhalation, transdermal (topical) and transmucosal administration), etc. The compositions may be administered alone, or in combination with any ingredient(s), active or inactive. Solutions or suspensions used for parenteral administration can include: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. A pharmaceutical composition can be aliquoted or packaged in ampules, disposable syringes, single or multiple dose vials made of glass or plastic, bottles, and the like, and such packaged forms, along with instructions for use, are within the scope of the present disclosure.

Pharmaceutical compositions adapted for injection include, for example, sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include, for example, physiological saline, bacteriostatic water, and phosphate buffered saline (PBS). The compositions prepared for administration should be sterile and should be fluid or convertible to a fluid at least sufficient for easy loading into a syringe. The composition and/or nucleic acid constructs should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi. Preservatives against microorganisms can include various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.

The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. 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 dispersions and by the use of surfactants.

In some instances, it may be desirable for the composition to be isotonic to blood. This can be accomplished using various isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.

Delayed or extended absorption of the injectable compositions can be achieved by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin, or by coating micro- or nano-particles of active agent in the composition with materials that delayed or extended release of components.

Sterile injectable solutions can be prepared, for example, by solubilizing or suspending the active compound in the required amount in an appropriate solvent with one or a combination of additional ingredients. Typically, creation of such solution or suspension is followed by sterile filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the other desired ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation is dried, e.g., by vacuum drying and/or freeze-drying.

Liposomal suspensions can also be used to prepare pharmaceutical compositions. These can be prepared according to methods known to those skilled in the art.

Oral or parenteral compositions can be formulated in dosage units for ease of

administration and uniformity of dosage. "Dosage unit form" as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

One of ordinary skill in the art will appreciate that the individual components of the present disclosure can change depending on the physical and chemical qualities needed for the pharmaceutical compositions in a given process and/or application to which the pharmaceutical compositions are applied.

Therapeutic Methods

In some aspects, the disclosure relates to methods of inhibiting serpin function or activity in a cell or a subject. Thus, in some embodiments, the disclosure provides a method for inhibiting serine proteinase activity in a cell, the method comprising delivering to the cell an isolated polypeptide ( e.g . an RCL peptide variant) as described herein, or a composition as described herein. A“subject” refers to a mammalian organism, for example a human, mouse, rat, cat, dog, hamster, Guinea pig, horse, etc. In some embodiments, a subject is a human or a mouse.

The disclosure is based, in part, on RCL peptide variants that exhibit increase activity (e.g., increased inhibition of certain serine proteases, such as thrombin) relative to currently availably RCL peptides (e.g., S-7). In some embodiments, an RCL peptide variant as described herein is at least 2-fold, 3-fold, 4-fold, 5-fold, lO-fold, 50-fold, lOO-fold, or lOOO-fold more effective in inhibiting a serine protease than S-7 peptide.

In some embodiments, methods described by the disclosure are useful for treating inflammatory diseases and disorders, for example unstable angina, vasculitis, heart attack, lupus, and certain types of inflammation caused by cancer and/or infection with a pathogen (e.g., a virus or bacteria). Accordingly, in some aspects, the disclosure provides an RCL peptide variant for use in treating a subject having or suspected of having an inflammatory disease or disorder.

The terms "treating" or "preventing" are used herein to mean the delay of the onset of one or more of the signs and/or symptoms, reducing the duration and/or severity of one or more of the signs or symptoms, reducing the number of symptoms, reducing the incidence of disease- related or infection-related symptoms, reducing the latency of symptoms, ameliorating symptoms, reducing any secondary symptoms, reducing any secondary infections, preventing relapse to a disease or infection, expediting remission, inducing remission, augmenting remission, speeding recovery, or increasing efficacy of or decreasing resistance to alternative therapeutics.

In some embodiments, a therapeutically effective amount of an RCL peptide variant or a composition ( e.g ., a composition comprising an RCL peptide variant) as described herein is administered to a subject. As used herein, the term "therapeutically effective amount" of a physiological or pharmaceutical composition refers to the amount that provides a treatment that delays, prevents one or more symptoms associated with a hemorrhagic infection or sepsis or even cures or ameliorates the infection or condition. Therapeutic administration also includes prophylactic applications. The RCL peptide variants described herein can also be used to prevent allograft rejection and treat and/or prevent allograft vascular disease.

The therapeutically effective amount of the compositions described herein and used in the methods disclosed herein applied to humans (e.g., patients, individuals, subjects) can be determined by one of ordinary skill in the art with consideration of individual differences in age, weight, and other factors, for example previous exposure to DNA virus or RNA virus or bacteria.

A physician can also choose a prophylactic administration wherein the subject (e.g., individual or patient) has an increased susceptibility (e.g., weakened immune system), a clinically determined predisposition or an increased risk to a hemorrhagic infection or sepsis. In therapeutic applications, compositions are administered to a subject (e.g., a human patient) already suffering from, for example, a hemorrhagic infection or sepsis in an amount sufficient to at least partially improve one or more signs or symptoms or to inhibit the progression of (and preferably arrest) the symptoms of the condition, its complications, and consequences.

Additionally, compositions can be administered to a patient undergoing a cardiac allograft transplant. Recovery can be accelerated in an individual who has been treated.

The present methods are effective for targeting thrombotic and thrombolytic pathways; reducing inflammation, lung consolidation, arterial inflammatory vasculitis and even colon dilatations well as bleeding (e.g., excess bleeding), and reducing clot formation. In some embodiments, methods described by the disclosure are useful for treating hemorrhagic viral or bacterial infections and viral or bacterial sepsis that is a result of a DNA or RNA virus or bacteria.

The methods of the present disclosure also include methods for treating a patient who has or is at risk for a hemorrhagic viral, bacterial or fungal infection. Further, the methods disclosed herein can include the treatment of transplant vascular disease including but not limited to cardiac allograft transplant in a patient. These methods can be carried out by, for example, administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising a serpin-derived peptide, or a biologically active variant or fragment thereof, as described herein. The methods can also include a step of identifying a patient in need of treatment ( e.g ., fever, severe headache, unexplained hemorrhage (bleeding or bruising)) and/or a patient who is at risk for a hemorrhagic viral infection (e.g., travel to an endemic country). The patient's symptoms (e.g., fever, severe headache, hemorrhage) can be associated with exposure to Ebola virus or Marburg virus.

In addition to administration of the compositions described herein, the patient can receive a second type of treatment for other infections. Examples of agents that can be used singly or in combination with the compositions described herein include but are not limited to anti-inflammatories (e.g., aspirin and interleukin 10), antibiotics, antivirals, clot inhibitors (e.g., heparin, anti-thrombin III, activated protein C, tissue factor pathway inhibitor, and

thrombomodulin), agents that block hemorrhage (e.g., factor VII), surgery, or any other treatment for sepsis or hemorrhage.

The disclosure is based, in part, on the discovery that certain RCL peptide variants described by the disclosure are therapeutically effective in subjects that have been administered antibiotic therapeutic agents. This is surprising in light of previous observations that RCL peptides, such as S-7, have reduced or no therapeutic activity in subjects having been administered antibiotics. Accordingly, in some aspects, the disclosure provides a method of inhibiting one or more serine proteases in a subject that has been previously administered an antimicrobial agent, the method comprising administering an RCL peptide variant described herein.

In some embodiments, a subject is administered one or more antimicrobial agents along with an RCL peptide variant as described herein. Lor example, a subject having a bacterial or viral infection may be administered, in some embodiments, one or more antimicrobial agents (e.g., antibiotic agents, antibacterial agents, antiviral agents, antiparasitic agents, etc.) in addition to an RCL peptide variant as described herein. The one or more additional therapeutic agents may be co-administered (e.g., as part of a single composition, or at the same time) or at different times (e.g., spatially or temporally distinct administration) as the RCL peptide variant. Other methods of the present disclosure are methods for treating a patient who has lethal sepsis (bacterial, viral or fungal), reducing inflammation and/or hemorrhage. The methods include a step of administering to a patient a therapeutically effective amount of a

pharmaceutical composition comprising a serpin-derived peptide, or a biologically active variant or fragment thereof, as described herein. The methods can also include a step to identify a patient in need of such treatment. Such patients include those who are suffering from a DNA or RNA virus, and those that have been exposed to or have a hemorrhagic viral infection caused by exposure to a filovirus ( e.g ., Ebola virus or Marburg virus). The filovirus genus includes Zaire Ebola virus, Sudan Ebola virus, Reston Ebola virus, Cote d'Ivoire Ebola virus and Marburg virus.

EXAMPLES

Examples of Serp-l reactive center loop (RCL) peptides, including S-l (I₃₁₇PRNAL₃₂₂), S-3 (R319NAL322), S-5 (T 323 AIV ANKPF331 ) , and S-7 (G305TT AS S DT AITLIPR319) are shown in FIG. 1. It has been observed that some of these peptides were poor inhibitors of uPA and tPA, and weak inhibitors for PAI-l, which is not inhibited by full-length Serp-l in vitro.

Additionally, it has been observed that after antibiotic treatment to suppress intestinal bacteria (the gut microbiome), RCL peptide activity was lost.

This Example describes Serp-l RCL peptide variants. In some embodiments, variant RCL peptides described by the disclosure have improved efficacy after antibiotic treatment in lethal MHV68 infections in mice.

Materials and Methods

Protein expression and purification

Recombinant Serp-l was expressed and harvested from Chinese Hamster Ovarian (CHO) cell line (Viron therapeutics Inc, London, ON, Canada). In some embodiments, Serp-l protein is estimated to be -95% purity as measured using Commas sie- stained SDS- polyacrylamide gels and reverse-phase HPLC.

Peptide synthesis and Mass spectrometry

Modified S-7 peptides and biotinylated S-7 peptide were obtained. For target identification, 50 pg (in 100 pL PBS) biotinylated S-7 peptide was loaded on a column with immobilized streptavidin. The target proteins from human and mouse (C57B1/6) plasma collected in heparin coated tubes were captured, washed and eluted. The eluted proteins were analyzed by mass spectrometry. The proteins were identified by Mascot (Matrix Science, London, UK; version 2.4.1). A control streptavidin column loaded with 100 pL PBS was also treated in the same way and the eluate from it was analyzed by LC-MS/MS to identify false positives.

Serp-1 Crystallization, data collection and processing.

High-throughput crystallization screening was performed on samples of purified Serp-l. Trays were incubated at room temperature and crystallization“hits” were observed after approximately 3 weeks. The screen that yielded the most“hits” was Natrix HT (HR2-131, Hampton Research, USA). A single Serp-l crystal was used for data collection and was obtained from the following crystallization condition: 0.2 M potassium chloride, 0.05 M magnesium chloride hexahydrate, 0.05 M Tris-HCl pH 7.5, 10% (w/v) polyethylene glycol (PEG) 4,000. X-ray diffraction data were collected at the Cornell High Energy Synchrotron Source (CHESS) on beamline Fl operating at 12.68 keV (l= 0.98 A). The data sets were collected using a Dectris Pilatus 6M detector at a crystal-to-detector distance of 350 mm with a 0.5° oscillation angle and an exposure time of 10 secs per image. A total of 360 images were collected. The data were indexed, integrated, and scaled using HKL2000. Data was indexed in the monoclinic C2 space group (unit cell dimensions a = 99.01, b = 122.1, c = 130.1 A, b = 94.9°), to a high resolution of 2.5 A, a completeness of 98.8%, and an R_sym of 4.1% (Table 1). Table 1

PDB accession # 6BJ5

Space Group C2

Cell Dimensions ( ;°) a = 99.01, b = 122.1, c = 130.1; b = 94.9

Resolution (A) 19.8-2.51 (2.57 - 2.51)

Total Reflections 53195

Unique Reflections 46146

Rsym^a (%) 4.1 (60.2)

Rpim^b(% ) 2.6 (37.8)

CCl/2 0.99 (0.81)

I/Is 9.2 (2.5)

Redundancy 3.4 (3.5)

Completeness (%) 98.8 (98.9)

Rcryst^C ( % ) 20.6 (26.3)

Rfree^d (%) 25.7 (35.5) VM (A³, Da ¹) 3.14

# of Protein Atoms 7879 (All Chains) # of Water Molecules 58

# of Ligand Atoms 20

Ramachandran stats (%): Favored, allowed, 96.7, 2.8, 0.5

generously allowed

Avg. B factors (A²): Main-chain, Side-chain, 43.7, 48.2, 30.8, 33.4

Solvent, Ligands

r.m.s.d. for bond lengths, angles (A,°) 0.010, 1.354

Model building, phasing, and refinements

No determined structure of Serp-l from Myxomavirus is listed in the Protein Data Bank. Therefore, initial phases were determined using molecular replacement with the structure of the homologous PAI-l (-35% sequence identity; PDB: 3LW2) with surface loops deleted and the core strands changed to Ala, as a search model. The poly- Ala PAI-l search model generated poor statistics from initial phasing, therefore Phenix AutoBuild was used to construct a more accurate model. Missing loops and atoms of Serp-l from the solution produced from AutoBuild were fitted manually using Coot. Structural refinements were carried out using Phenix with 5% of the unique reflections excluded to calculate Rfr_ee. Manual refitting of the model between each refinement and construction and fitting of ligands was done using Coot. The final model of Serp-l was refined to an Rcryst of 20.6 and Rf_ree of 25.7% (Table 1). The model geometries and statistics were assessed by PROCHECK. All figures were made using PyMOL (Schrodinger LLC).

Peptides

Free energy calculations were performed using GROMACS. The proteins and peptides were modeled with the AMBER ff-03 force field and the TIP3P model described water molecules. Protein structures were taken from the PDB for ATIII (PDB: 1ATT) and PAI-l (PDB: 3CVM). All calculations were done in a periodical box of water with dimension

60x60x60 A. Free energy perturbation (FEP) calculations were done using the Thermodynamic Integration and Umbrella Sampling/WHAM methods. These calculations have error bars on the order of ±1 kcal/mol.

Molecular docking and modeling of RCL peptides

Docking and modeling software were utilized to determine the binding potential of RCL- based peptides from Serp-l with target proteins, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA), and human serpins PAI-l, ATM, and A1AT to explore a potential rationale for the observed immunomodulatory effects of Serp-l and to implement a strategy for designing improved RCL peptides. Models of peptides, or peptide- serpin complexes, used for docking experiments were made using Coot. RCL peptide

coordinates were established by utilizing similar designated regions of the RCL corresponding to the interfaces between uPA/tPA and PAI-l (PDBs: 5BRR and 3PB1). These coordinates acted as a starting point in determining binding energies of RCL peptides with target proteases. RCL peptides S-7, MPS7-8 and MPS7-9 were modeled as b-strands in inactive (with cleaved RCLs) forms of PAI-l (PDB: 1A7C), ATM (PDB: 2B4X), and A1AT (PDB: 4PYW) to predict binding modes of each peptide for rationale peptide design. Surface area (A²) and estimated number of hydrogen bonds formed were estimated using PDBe software, Protein Interfaces, Surfaces, and Assemblies (PISA) and presented in Tables 2 and 3. Auto Dock Vina software was utilized to predict binding energy of peptide/serpin interfaces (AGi_nt) in attempts to predict improved affinity of peptides with target serpins. Utilizing the structure of Serp-l, and an overlay of S-7 coordinates with other target serpins to estimate a force-field size for sampling of low-energy conformations of peptides-serpin complexes. Hydrogens, but not solvent molecules, were used in calculations of predicted peptide coordinates and binding energies. Results are summarized in Tables 2 and 3.

Table 2

_ uPA _ tPA _

RCL Peptide Origin AGi_nt (k _{ca i}/mol) Interface Surface AGi_m Interface Surface _ Area (A²) _ (k_Cai/mol) _ Area (A²)

Serp-l Ϊ 526.2 GA 546.0

PAI-l -4.9 504.7 -5.3 521.9

Table 3 S-7 Residue (position) Stabilizing mutations: Stabilizing mutations: Stabilizing

ATIII PAI-1 mutations: ATIII

Modeling ofSerp-1 glycosylation

Electron density for glycans associated with Asn 28 glycosylation was observed, however not for Asn 99 or 172 (FIG. 12). This is most likely due to the highly flexible nature of the A- linked glycan causing a reduction in signal from the electron density map. Further, through observations of the crystal packing of Serp-l, surface glycan most likely exists in the unoccupied space between asymmetric units (AUs) in the unit cell, allowing for a high range of mobility and thus a loss of signal in the electron density (FIG. 9). Despite the lack of well- ordered density for most of the glycosylation, previous mass spectrometry analysis has confirmed that Asn 28, 99, and 172, are fully glycosylated upon Serp-l production. GlyProt, was utilized to model in full-length glycosylation chains on Asn 28, 99, and 172 (FIG. 12). Glycsolylation was modeled based on geometry and appeared to occupy the“vacant space” located between proteomeric units of Serp-l within the unit cell (FIG. 9). Fluorogenic real-time kinetic assay

The reduction of thrombin activity by ATIII (ThermoFisher #RP-43l36) was measured using the SensoFyte 520 Thrombin Activity Assay kit (Anaspec). Protocols were performed according to manufacturer’s instructions, with the following modifications. For the thrombin activity assay, ATIII was added at 1:1 molar equivalence in the presence of 2- fold excess heparin. Peptides were pre-incubated with the ATIII for 10 minutes at 37 °C prior to addition of proteases. Plates were read on a Molecular Devices M5e multi-mode plate reader with 490 nm excitation and 520 nm emission every 30 seconds for 60 minutes.

SDS-PAGE of Thrombin- ATIII complex formation

ATIII (100 ng; ThermoFisher #RP-43136) was pre-incubated with respective peptides (1 pg) for 10 minutes at 37 °C prior to addition of thrombin (300 ng; ProSpec #PRO-l422) and subsequent incubation at 37 °C for an additional 20 minutes in a total volume of 12 pF. Samples were loaded with native loading buffer (Morganville Scientific #LB0200) and resolved on a 12% SDS-PAGE. Resolved gels were stained with SimplyBlue SafeStain (ThermoFisher) according to manufacturer’s instructions (Thermo Fisher). Images were collected on a GE FAS4000 with the high resolution setting and 2 seconds of exposure by transillumination in the third tray position.

RCL-derived peptide survival assays

Survival assays using MHV68 infection in IFNyR ⁷ mice were performed. MHV68 stocks were generated as previously described in NIH 3T12 fibroblasts (ATCC, Manassas, VA) infected with an MOI of 0.1 at 50% confluency. Seven days post-infection cells were frozen and thawed and lysate transferred to Nalgene Oak Ridge PPCO tubes and centrifuged (l5min at 4300 x g ). Supernatant was centrifuged (2 h at 12,000 x g ) and the pellet rinsed with PBS, re suspended in media, vortexed, and stored at -80 °C in 250 pl aliquots. Virus was titered in duplicate. Antibiotic solutions used for treatments were made fresh weekly in autoclaved water with Streptomycin (2gm/F), Gentamicin (0.5gm/F), Bacitracin (lgm/F), and Ciprofloxacin (0.l25gm/F). The bottles were changed out after 7 days with freshly made antibiotics solution. Treatment duration was 10 days. On the 1 I^th morning, the water bottles with antibiotic were exchanged for autoclaved water and the mice were infected with MHV68 (I.P.) followed by daily treatment with one of the peptides or saline (I.P.).

Data

Serp-1 structure and crystallographic arrangement

The structure of Serp-l was determined in a C2 space group (unit cell dimensions of a = 99.01, b = 122.1, c = 130.1 A; b = 94.9°) to a high resolution of 2.5 A, and an Rcryst and Rfree of 20.6 and 25.7 %, respectively (Table 1). For the overall structure of Serp-l the protein was observed to be in a cleaved, inactive form, similar to that observed in serpins derived from other organisms. The overall structure of Serp-l was compared to human serpins PAI-l (PDB:

1A7C), ATIII (PDB: 2B4X), and A1AT (PDB: 4PYW). The comparison of Serp-l to human serpins was utilized to determine structure-function relationships between each protein. It was observed that Serp-l shows homology with three human serpins, in cleaved forms with comparable Cas r.m.s.d. values of 1.0, 1.7, and 1.8 A for PAI-l, ATIII, and A1AT, respectively (FIG. 2 and FIG. 8). The highest degree of variability between Serp-l and human serpins appears toward the surface, with a general conservation of core structure observed between each protein (FIG. 2).

It was observed that the crystal packing of Serp-l displays large‘gaps’ located between each asymmetric unit (AU), which consists of non-biological trimers (as Serp-l is monomeric in solution) (FIG. 9). It was also observed that Serp-l is heavily glycosylated with L inked glycosylation at Asn 28, 99, and 172. Analysis of these glycosylation sites on Serp-l within the unit cell, shows that these spaces may be occupied by highly flexible high mannose-type arrangements, thus providing a further explanation to the apparent“loosely” packed unit cell (FIG. 9). In addition, a molecule of polyethylene-glycol (PEG) located between crystallographic monomers of Serp-l was observed in the electron density (FIG. 9). The presence of PEG, which is in the crystallization condition, indicates it (or a similar agent) is necessary to induce Serp-l crystallization, as it was observed to act as a scaffold between symmetry related units of Serp-l within the unit cell of this space group.

Binding regions and predicted functionality of RCL-derived peptides

The Serp-l RCL typically consists of an area on the protein from residues 305 - 331.

The crystal structure of Serp-l was utilized to‘map’ important binding regions of each peptide and provide a rationale for mechanisms of action exhibited by these RCL-derived peptides. For example, S-7, which is derived from RCL residues 305 - 319, was tested. In the structure of cleaved Serp-l presented, the positions of residues associated with S-7 in Serp-l are shown to form a b-strand at the core of the protein, and appear predominantly stabilized by main-chain hydrogen bonding. A similar binding mode of S-7 with target serpins is shown via an overlay of the coordinates of the S-7 peptide from Serp-l with PAI-l (highest structural homology with Serp-l) where the positions of residues are highly conserved (FIG. 3). This is also true when comparing coordinates of residues of S-7 with positions in ATIII and A1AT further indicating S-7 may act by inserting into serpins as a b-strand leading to inactivation where the peptide becomes an integral part of the b-structure at the core of the protein.

To test potential for RCL peptide, S-7, binding with other serpins in the circulating blood, mass spectrometry using both human and mouse plasma was performed. Data indicate that S-7 potentially interacts directly with both human and mouse serpins, specifically ATIII and A1AT in mouse plasma and ATIII in human plasma (Table 4). A full list of proteins pulled down on MS/MS is provided in Table 5. This result, coupled with the structural information presented, indicates a correlation between S-7 inactivation of human serpins resulting in the observed therapeutic effect during S-7 administration. This further indicates that insertion of S- 7 into human serpins translates directly to some of the observed therapeutic effects seen through its administration and indicates a target site for RCL peptide designs (FIG. 3 and Table 4).

5

Table 4

MS/MS Proteins bound to S- Accession Number MW (kDa) Plasma Unique 7 Peptide Detected by MS _ Peptides

Antithrombin-III (ATIII)^a A0A024R944_HUMAN (+1) 53 H. sapiens 7

Inter-alpha-trypsin inhibitor¹³ A0A087WTE1_HUMAN (+4) 107 H. sapiens 8

Antithrombin-III (ATIII)^a ANT3_MOUSE (+1) 52 M. musculus 5

Alpha- 1 -antitrypsin (AlAT)^a _ A1AT2_MQUSE (+4) _ 46 _ M. musculus _ 2 ^aSerpin

b Inter-alpha inhibitor protein (IAIP) serine protease inhibitor

Table 5

Serp-l, PAI-l, and ATIII all share a similar R-Pl at the R1-RG scissile bond in the RCL, enabling cleavage and binding to the targeted serine proteases ( e.g . tPA, uPA plasmin and thrombin, and for Serp-l, FXa), whereas in A1AT there is an M-Pl. Amino acids of Serp-l corresponding to S-l and S-3 in the RCL of the native (uncleaved; PDB: 5BRR), latent

(transition state prior to cleavage; PDB: 1LJ5) and cleaved (PDB: 1A7C) structures of PAI-l (used as a reference for human serpins; FIG. 4), were not present due to cleavage prior to, or during, crystallization. Structures of PAI-l :uPA (PDB: 3PB1) and PAI-l :tPA (PDB: 5BRR) show interactions occurring directly with the RCL. Due to the high structural homology between Serp-l and the human serpins (FIG. 2), Serp-l likely interacts with uPA/tPA, and other protease targets similarly. In addition, both uPA and tPA are implicated in plasminogen activation and fibrinolysis as well as activation of matrix degrading metalloproteases with their inhibition being important for regulation of these processes. Therefore, the therapeutic effects observed when administering S-l or S-3 may occur through uPA/tPA inhibition. Despite this, it has been observed that weak inhibition of uPA/tPA is exhibited by S-l and S-3 via the observed binding regions of S-l and S-3, which occupy a shallow binding pocket on uPA/tPA with limited apparent stabilizing interactions (FIG. 4). In addition, R-Pl, which is necessary for forming the Michaelis serpin-protease complex, is located very close to, or on, the N-terminus of both S-l and S-3, respectively. In addition, in silico modeling using PDBe PISA of the S-l and S-3 peptides, along with corresponding residues from PAI-l, show comparable interface binding energies further indicating that observed inhibition of uPA/tPA from full-length Serp-l occurs through the RCL in combination with proximal interactions (Table 2).

S-5 showed the least therapeutic potential compared to the other RCL-derived peptides. This can be explained by observing the positions occupied by residues of the RCL

corresponding to S-5 in Serp-l and PAI-L In the cleaved form of Serp-l, residues

corresponding to S-5 are observed occupying a shallow pocket towards the surface of the serpin (FIG. 10). Residues in this region also appear to be highly flexible (B-factors of Cas >60 A²), and thus are likely to have low affinity due to a reduction in stabilizing interactions. In addition, R-Pl is not present on S-5, which can also play a role in the limited interactions with both target proteases and serpins, such as PAI-l. This further indicates difficulties in targeting this region in PAI-l or other human serpins, for example the observed reduction in therapeutic efficacy when administered (FIG. 10).

Free energy changes associated with single point mutants to S-7 (15 amino acids in RCL peptides of Serp-l) were calculated and the following criteria were implemented for peptide design: 1) implementing residue substitutions toward the C-terminus (facing toward the protein surface) of the peptide in order to minimize potential loss of binding interactions due to steric hindrance of residue side-chains in the protein core, and 2) increasing the potential for hydrogen bonding interactions in regions that are conserved or semi-conserved between each of the human serpins. A region surrounding a conserved Thr (positions 161, 211, and 180 in PAI-l, ATIII, and A1AT, respectively), and a semi-conserved position that contains an Asp in PAI-l and A1AT (positions 158 and 177, respectively), and an Asn in ATIII (position 208) (FIG. 5 and FIG. 8) were investigated.

Free Energy Perturbation (FEP) calculations using the Thermodynamic Integration (TI) method were used to compute the difference in free energy (AAG) between S-7 and mutated peptides, where negative (AAG) values indicate the increase in binding affinity of the peptide to the human serpins. This value is a combination of the peptide states, both in solvent as well as bound to the protein. Initial structures of complex of S-7 with ATIII and PAI-l serpins can be found in FIG. 11.

The results of these calculations, indicated several mutations that to stabilize (have stronger binding) the mutated peptide-serpins complexes (Table 3). Negatively charged residue substitutions, Glu and Asp (pK_a of 4.07 and 3.86, respectively), were favorable in order to increase the likelihood to form stabilizing hydrogen bonds with either adjacent residues or solvent at physiological pH. As such, the resultant peptides that displayed the highest favorable binding to human serpins were: MPS7-8 (G305TTASSDT AITLEPR319) (SEQ ID NO: 2), containing a single Ile to Glu substitution, and MPS7-9 (G305TTASSDTAITDEPR319) (SEQ ID NO: 3) that has two substitutions of Ile to Glu and Leu to Asp (FIG. 1).

MPS7 binding to human serpins

Once FEP results identified the two S-7 derivative peptides, MPS7-8 and MPS7-9, they were further tested in-silico using the determined Serp-l crystal structure. Modeling S-7 into each of the human serpins indicated a conserved binding region where S-7 acts as a substitute for the cleaved RCL that forms the b-strand at the core resulting in an inactive serpin complex (FIG. 3). The same procedure was repeated to model MPS7-8 and MPS7-9 into PAI-l, ATIII and A1AT. Data indicate the C5 of Glu in both MPS7-8 and MPS7-9 resides is <3 A from the CP of the conserved Thr, which is within potential hydrogen bonding distance.

Compared to S-7, which contains an Ile in this position, the distance increases to >3 A between similar atoms (FIG. 5). A similar observation was made where the side-chain of Glu in both peptides is within hydrogen bonding distances (<3.5 A) of Asp 158 and 177 in PAI-l and A1AT, respectively, and Asn 208 in ATIII. When considering Ile of S-7, this distance increases to >4 A, which may result in drastic loss of interactions between these residue positions (FIG.

5). The Leu to Asp substitution in MPS7-9 facilitates potential hydrogen bonding interactions between the Asp side-chain, and main-chain atoms of Val 214 and 311 in ATIII and A1AT, respectively, and Arg 162 and Leu 163 in PAI-L This is observation is based on the estimated <3 A distance observed between the Cy of Asp in MPS7-9 and main-chain atoms of the aforementioned residues (FIG. 5). Thus, in some embodiments, the Asp substitution further induces more hydrogen bonding interactions resulting in more favorable binding to human serpins.

PDBe PISA modeling software was utilized to estimate number of hydrogen bonds formed, and Autodock Vina docking software was used to estimate binding potential of each peptide to the human serpins through free energy calculations (Table 6). For all human serpins tested, there was an increase of hydrogen bonds formed in MPS7-8 and MPS7-9 compared to S- 7. In addition, a noticeable decrease in hydrophobic interface interactions in MPS7-8 and

MPS7-9 compared to S-7 was observed, shown through estimations of AGi_nt from PDBe PISA results (Table 6). In this case, AGi_nt is an estimation of favorable hydrophobic interactions formed between interfaces of ligands and protein surfaces, whereby a larger negative number indicates an increase in hydrophobic surface interactions. A clear trend where AGi_nt becomes increasingly positive when compared from S-7 to MPS7-8 and MPS7-9 was observed in all cases, further indicating an increase in hydrophilic interactions with Glu, or Glu and Asp substitutions (Table 6). When considering polar interactions through Autodock Vina , the trend reverses indicating an increase in RCL peptide affinity with human serpins in MPS7-8 and

MPS7-9 compared to S-7 (Table 6). Table 6

S-7 MPS7-8 MPS7-9

PAI-1

AGint (k ca i/mol) -l2.4(-5.0) l0.8(-5.5) -8.9(-6.3) Interface Surface Area (A²) 1794 1787 1780

Num. of H-bonds 29 31 32

ATIII

AGint (k ca i/mol) -l l.7(-5.7) -9.2(-6.6) -7.5(-7.0) Interface Surface Area (A²) 1801 1794 1770

Num. of H-bonds 20 22 24

A1AT

AGint (k ca i/mol) -l2.4(-5.2) T0.7(-5.3) -9.0(-5.4) Interface Surface Area (A²) 1791 1775 1772

Num. of H-bonds 25 26 28

In vitro binding analysis of S-7, MPS7-8, and MPS7-9 with target serpins and proteases

Based on in silico analysis that the RCL peptides may function by binding to circulating serpins and target proteases, the capacity of S-7, MPS7-8, and MPS7-9 to interfere with serpin- protease binding was investigated. Specifically, the capacity for each peptide to block thrombin to ATIII was assessed based upon thrombin as a known target of Serp-l. Kinetic thrombin- protease fluorogenic assays were unable to detect inhibitory effects of S-7, MPS7-8, or MPS7- 9, on serpin inhibition of protease activity (FIG. 6). Non-reducing SDS-PAGE indicated an appreciable interference of thrombin- ATIII complex formation as indicated by an increase in unbound ATIII (FIG. 6). The detectable interference of complex formation in the absence of kinetic changes indicates that the therapeutic efficacy of the S-7, MPS7-8, and MPS7-9 peptides is primarily an emergent phenomenon that is most readily detectible by in vivo assay.

MPS7-8 and MPS7-9 demonstrate increased therapeutic effect compared to S-7

It has been observed that the beneficial serpin function of S-7 administration was lost in MHV68-infected IFNyRKO mice after antibiotic treatment with suppression of gut bacteria (FIG. 7; N=20 mice; P=NS). The modified serpin peptides, MPS7-8 and MPS7-9, were observed to partially restore therapeutic efficacy after antibiotic treatments with a demonstrated improved survival in this model (N=l0 mice; P<0.000l) (FIG. 7). This result was striking as simple non-polar to polar residue substitutions on MPS7 peptides were capable of increasing and restoring, in part, the beneficial therapeutic effect for the S-7 RCL peptide treatments that was lost in the presence of antibiotic treatments. In silico and in vitro data coupled with the observations presented from these survival assays (FIG. 7) indicate that the affinity of RCL peptides towards human serpins was increased and therapeutic potential improved.

Observed glycosylation of Serp-l

Serp- l exists as a heavily glycosylated protein that has three AM inked glycosylation sites at Asn 28, 99, and 172 (FIG. 12). Glycosylation at Asn 99 and 172 has been observed to be important for Serp-l production, with Asn 172 and 99 being important for secretion regulation. Currently, the function of glycosylation at Asn 28 is unknown. In some embodiments, the presence of glycosylation in Serp- l at Asn 28, is important for protecting the protein against proteolysis and formation of inactive aggregates. In the crystal structure of Serp-l, limited electron density is observed for each glycosylation site with the most prominent being observed as Asn 28 (FIG. 12). Nonetheless, due to the‘loose’ crystal packing of AUs, in combination with previous mass spectrometry data, indicate that the glycosylation is most likely present in the Serp-l crystal structure (FIG. 12). Therefore, to show the structure of fully glycosylated Serp- l, GlyProt, which models glycosylation of select residues through evaluation of residue type and likelihood for glycosylic modification, spatial accessibility, chemical geometry of the predicted glycan, and possible physicochemical properties was used to investigate glycosylic arrangements (FIG. 12).

SEQUENCES

>SEQ ID NO: l ; Myxomavirus Serp- l ( e.g . , UniProtKB/Swiss-Prot: P12393.2)

MKYLVLVLCLT S C ACRDIGLWTFR Y V YNES DN V VF S P Y GLT SALS VLRIA AGGNTKREI D VPES V VEDS D AFLALRELF VD AS VPLRPEFT AEFS S RFNT S V QRVTFN S ENVKD VIN S Y VKDKTGGDVPRVLDASLDRDTKMLLLSSVRMKTSWRHVFDPSFTTDQPFYSGNVTYK VRMMNKIDTLKTETFTLRN V GY S VTELP YKRRQT AMLLV VPDDLGEIVR ALDLS LVRF WIRNMRKD VCQ V VMPKF S VES VLDLRD ALQRLG VRD AFDPS RADFGQ AS PS NDLY VT KVLQT S KIE ADERGTT AS S DT AITLIPRN ALT AIV ANKPFMFLIYHKPTTT VLFMGTITKG EKVIYDTEGRDD V V S S V

EQUIVALENTS

Having thus described several aspects of at least one embodiment of this invention, it is to be appreciated that various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and scope of the invention.

Accordingly, the foregoing description and drawings are by way of example only.

While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or

configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and/or methods, if such features, systems, articles, materials, and/or methods are not mutually inconsistent, is included within the scope of the present invention.

The indefinite articles“a” and“an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean“at least one.”

The phrase“and/or,” as used herein in the specification and in the claims, should be understood to mean“either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to“A and/or B,” when used in conjunction with open-ended language such as“comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc. As used herein in the specification and in the claims,“or” should be understood to have the same meaning as“and/or” as defined above. For example, when separating items in a list, “or” or“and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as“only one of’ or“exactly one of,” or, when used in the claims,“consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term“or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e.“one or the other but not both”) when preceded by terms of exclusivity, such as“either,”“one of,”“only one of,” or“exactly one of.”“Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase“at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase“at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example,“at least one of A and B” (or, equivalently,“at least one of A or B,” or, equivalently“at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

In the claims, as well as in the specification above, all transitional phrases such as “comprising,”“including,”“carrying,”“having,”“containing,”“involving,”“holding,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases“consisting of’ and“consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. Use of ordinal terms such as“first,”“second,”“third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements. It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

Claims

CLAIMS What is claimed is:

1. An isolated polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of Myxoma virus Serine Proteinase Inhibitor 1 (Serp-l) as provided in SEQ ID NO: 1, wherein the amino acid sequence of the recombinant protein comprises one or more mutations at position L316, 1317, or L316 and 1317.

2. The isolated polypeptide of claim 1, wherein the isolated polypeptide is at least 95% or at least 99% identical to the amino acid sequence as set forth in SEQ ID NO: 1.

3. The isolated polypeptide of claim 1 or 2, wherein the one or more mutations are L316D, I317E, or L3l6D and I317E.

4. The isolated polypeptide of any one of claims 1 to 3, wherein the isolated polypeptide comprises the sequence set forth in SEQ ID NO: 2 or 3.

5. The isolated polypeptide of any one of claims 1 to 4, wherein the isolated polypeptide inhibits activity of one or more serine proteinase, optionally wherein the serine proteinase is a human serpin.

6. An isolated polypeptide comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 4 (GTT AS S DT AITX i X₂PR) ;

wherein if Xi is leucine, then X₂ is not isoleucine; and

wherein if X₂ is isoleucine, then Xi is not leucine.

7. The isolated polypeptide of claim 6, wherein Xi is any polar amino acid.

8. The isolated polypeptide of claim 6, wherein X₂ is any polar amino acid.

9. The isolated polypeptide of claim 6, wherein Xi is any charged amino acid.

10. The isolated polypeptide of claim 6, wherein X₂ is any charged amino acid.

11. The isolated polypeptide of claim 6, wherein X₂ is glutamic acid, optionally wherein the isolated polypeptide comprises the sequence set forth in SEQ ID NO: 2.

12. The isolated polypeptide of claim 6, wherein Xi is aspartic acid.

13. The isolated polypeptide of claim 6, wherein Xi is aspartic acid and X₂ is glutamic acid, optionally wherein the isolated polypeptide comprises the sequence set forth in SEQ ID NO: 3.

14. The isolated polypeptide of any one of claims 6 to 13, wherein the isolated polypeptide inhibits activity of one or more serine proteinase, optionally wherein the serine proteinase is a human serpin.

15. An isolated nucleic acid comprising a nucleic acid sequence encoding the isolated polypeptide of any one of claims 1 to 14.

16. A composition comprising the isolated polypeptide of any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

17. A method for inhibiting serine proteinase activity in a cell, the method comprising delivering to the cell the isolated polypeptide of any one of claims 1 to 14, or the composition of claim 16.

18. The method of claim 17, wherein the cell is a mammalian cell, optionally a human cell or a rodent cell ( e.g ., mouse cell).

19. A method for treating an inflammatory disorder or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of the isolated polypeptide of any one of claims 1 to 14, or the composition of claim 16.

20. The method of claim 19, wherein the inflammatory disorder or condition is vasculitis, optionally vasculitis characterized by lung hemorrhage, lupus, viral sepsis, or transplant rejection.

21. The method of claim 19 or 20, wherein the subject is a mammal, optionally wherein the subject is a human or a rodent ( e.g ., a mouse).

22. The method of any one of claims 19 to 21, wherein the subject has been previously treated with an antibiotic agent.

23. The method of claim 22, wherein the subject is characterized by a suppression of gut bacteria.