阅读说明：本技术 通用抗毒液素 (Universal antitoxic liquid ) 是由 允·Y·黄 于 2018-02-16 设计创作，主要内容包括：本公开涉及一种用于治疗有毒动物咬伤的通用抗毒液素,以及使用新型靶定的噬菌体展示技术开发所述通用抗毒液素的方法。(The present disclosure relates to a universal antitoxin for use in the treatment of venomous animal bites, and methods for developing the universal antitoxin using novel targeted phage display technologies.)

1. A multi-species antitoxin composition comprising a bacteriophage-expressed peptide that binds to a target common to more than one animal venom; and a pharmaceutically acceptable carrier.

2. The composition of claim 1, wherein the phage-expressed peptide is about 7 to about 12 amino acids in length; and wherein the target is a metal, a carbohydrate, a protein, or any combination thereof.

3. The composition of claim 1 or 2, wherein the peptide is linear.

4. The composition of claim 1 or 2, wherein the peptide is cyclic.

5. The composition of any one of claims 1-4, wherein the target is a protein.

6. The composition of any one of claims 1-5, wherein the target is phospholipase A₂(PLA₂)。

7. The composition of any one of claims 1-6, wherein the peptide is selected from the group consisting of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, and any combination thereof.

8. The composition of claim 7, wherein the peptide comprises SEQ ID NO 1.

9. The composition of claim 7, wherein the peptide comprises SEQ ID NO 2.

10. The composition of claim 7, wherein the peptide comprises SEQ ID NO 3.

11. The composition of claim 7, wherein the peptide comprises SEQ ID NO 4.

12. The composition of any one of claims 1-11, wherein the peptide is selected from the group consisting of SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, and any combination thereof.

13. The composition of claim 12, wherein the peptide comprises SEQ ID NO 9.

14. The composition of claim 12, wherein the peptide comprises SEQ ID NO 10.

15. The composition of claim 12, wherein the peptide comprises SEQ ID NO 11.

16. The composition of claim 12, wherein the peptide comprises SEQ ID NO 12.

17. The composition of any one of claims 1-16, wherein the phage-expressed peptide is expressed by a M13 phage.

18. The composition of any one of claims 1-17, wherein a plurality of animal venom is derived from one or more snake species.

19. A bacteriophage-expressed peptide that binds to a target in one or more animal venom.

20. The phage-expressed peptide of claim 19, wherein said peptide is selected from the group consisting of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, and any combination thereof.

21. The phage-expressed peptide of claim 19 or 20, wherein said target is phospholipase a₂。

22. The phage-expressed peptide of any one of claims 19-21, further comprising a peptide selected from the group consisting of SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, and any combination thereof.

23. The phage-expressed peptide of any of claims 19-22, wherein said phage-expressed peptide is expressed by a M13 phage.

24. An isolated nucleic acid molecule encoding the peptide of any one of claims 19-23.

25. The nucleic acid molecule of claim 24, comprising a sequence selected from the group consisting of SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, and any combination thereof.

26. A vector comprising a nucleic acid encoding the peptide of any one of claims 19-23.

27. A vector comprising the nucleic acid molecule of claim 24 or 25.

28. A host cell comprising the isolated nucleic acid molecule of claim 24 or 25, or the isolated vector of claim 26 or 27.

29. The host cell of claim 28, which is a prokaryotic cell.

30. The host cell of claim 29 which is e.

31. The host cell of claim 30 which is e.coli K12ER 2738.

32. A method of producing the composition of any one of claims 1-18 or the peptide of any one of claims 19-23, comprising culturing the host cell of any one of claims 28-31 under conditions to produce the peptide.

33. A method of producing the composition of any one of claims 1-18 or the peptide of any one of claims 19-23, comprising:

a. confirmation of common PLA in Western cottony Agkistrodon Halys venom₂A protein sequence;

b. identification of consensus PLA in other snake species₂A protein sequence; and

c. one or more phage are produced via phage display panning.

34. A method of treating a subject in contact with animal venom comprising administering to the subject the composition of any one of claims 1-18.

35. The method of claim 34, wherein said animal venom is from western cottontopodium venom.

36. The method of claim 34 or 35, wherein said composition binds said animal venom, thereby neutralizing venom toxicity.

37. The method of any one of claims 34-36, wherein the composition comprises a peptide selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, and any combination thereof.

38. The method of any one of claims 34-37, wherein the composition is administered orally, rectally, transdermally, intravenously, intramuscularly, intraperitoneally, intramedullally, epidurally, or subcutaneously.

39. A diagnostic kit comprising the composition of any one of claims 1-18.

40. The kit of claim 39, wherein the composition comprises a peptide selected from the group consisting of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, and any combination thereof.

41. The kit of claim 39 or 40, wherein the composition comprises a peptide selected from the group consisting of SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, and any combination thereof.

Technical Field

The present disclosure relates generally to universal antitoxin (antiphom) development using phage-displayed short peptides.

Background

M13 phage expressing short peptides have been used as delivery vehicles (delivery vehicles) to transport various binding motifs to a target. Genetic modification of the phage tail protein allows the expression of unique peptides with variable sequence, length and composition. The expressed peptides can bind to specific epitopes, thus forming the basis of a high-throughput system for the identification of binding partners.

Animal venom sting (envenomation) is a major public health problem worldwide and is classified as an overlooked disease by the world health organization. For example, about 40 million people worldwide, with nearly 9,000 people per year in the united states and canada affected by venomous snake bite venom stings. Several antibody-based antitoxic agents are available for enteral therapy. Multivalent immune Fab (ovine) of Crotalus is the only product which can be widely used for treating patients with Crotalus; however, its high cost and side effects are common problems.

Antibody-based antitoxicins were developed by exposing host animals to neat venom for immunomodulation and extracting the resulting antibodies. Although antibody-based strategies have resulted in successful therapies against certain snake species, there are still limitations on the safety, efficacy and cost of manufacture. In addition, antibody-based strategies have limited effectiveness against venom stings from other animals (arachnids and jellyfish).

Serum was then separated from the animals and venom reactive antibodies were purified. Although this antibody-based strategy has resulted in successful therapy against certain snake species, limitations remain in terms of safety, efficacy and economics of manufacture. One of the most serious side effects of antibody-based antivenins is the immune response of the patient to heterologous immunoglobulins from horses or sheep, known as seropathy. Furthermore, most antibody-based solutions either require special storage conditions or, in the case of lyophilization, require reconstitution prior to administration; both of which reduce their utility in remote and severe conditions. While others have produced antibody-based antitoxicins, their continued pursuit for antitoxic production is questionable in view of the impact of expensive and time-consuming production processes and limitations of application.

The following references provide background information on the prior art of antitoxin technology and are incorporated herein by reference in their entirety: molenaar, T.J. et al, Uptake and processing of modified bacteriophageM13 in microorganisms for phase display.virology 293,182-191, doi-0.1006/micro.2001.1254 (2002); rabies and Envenomings A New selected Public Health Issue (WHO 2007); WHO guides for the Production Control and Regulation of Snake antibacterial immunolobulins (WHO 2010); warrell, d.a. guidelines for the management of snake-bits (WHO 2010); smith, S. et al, Bendside management associations inter stream treatment of pit view evolution. J Emerg Nurs 40,537-545, doi: l0.1016/j. jen.2014.01.002 (2014); mowry, J.B., Spyker, D.A., Cantilena, L.R., Jr., Bailey, J.E. & Ford, M.2012Annual Report of the American Association of position controls' National Position Data System (NPDS):30th annular Report. clin. toxicol (Phila)51,949-1229, doi:10.3109/15563650.2013.863906 (2013); wilderness Medical Society practices Guidelines for the Treatment of Pitviperver Engineers in the United States and Canada. Wilderness Environ Med 26,472 487, doi: l0.1016/j. wem.2015.05.007 (2015); holland, D.R. et al, The crystallization structure of a lysine 49 phosphonase A2 from The evolution of The cottonmohsNAke at 2.0-A resolution, J Biol Chem 265,17649-17656 (1990); fralick, J., Chadha-Mohanty, P. & Li, G.in Advances in Biological and Chemical Terrorism counter easureres (eds R.Kendall, S.Presley, G.Austin, & P.Smith)179- & 202(CRC Press, 2008); philipson, L., Albertsson, P.A., Frick, G.the publication and control of viruses by aqueous polymer phase systems, virology,11,553-571 (1960); yu, J. & Smith, G.P. [1] Affinity matching of phase-displayed peptide ligands, 267,3-27, doi: 10.1016/s 0076-6879(96), (67003-7), (1996); prakash s.s.phage display technology for anti-vector production. clinical Microbiology and Infection 13:4(October 2015); phancolato, E.C. et al, Phage display as a novel simulating anti-infective herepy: a review.93:79-84 Toxicon (Jan.2015; Epub Nov.2014).

Disclosure of Invention

The present disclosure provides a multi-species antitoxic liquiritin composition. The compositions contain phage-expressed peptides that bind to a target common to several different animal venoms. The phage-expressed peptides are typically 7-12 mer peptides that can bind to a number of targets, including metals, carbohydrates, and proteins.

The present disclosure also provides an improved method of peptide target design based on editing consensus protein sequences (consensus) reflecting multiple animal venoms. The consensus protein sequence then provides a target for phage display panning (affinity partitioning of enriched homologous regions to different venom protein sequences).

At one endIn exemplary embodiments, the present disclosure provides a Western cottontomate venom based on consensus phospholipase a in Western cottontomate venom₂(PLA₂) Improved methods for peptide target design of protein sequences. Thus, the consensus sequence provides a target for phage display panning in the generation of an antidote that is universal to north american vipers. The method includes redefining the target sequence to include homologous regions of the seven most common vipers in north america. In particular, the method involves redefining the target peptide by targeting a conserved active site to mimic the homologous region of the peptide family. The method may use phage-expressed peptides listed in table 1 below.

The present disclosure also relates to an antitoxic formulation (formulation) comprising a phage-expressed peptide suspended in a pharmaceutically acceptable carrier (carrier), wherein the peptide is configured to bind to a conserved snake venom component, thereby neutralizing venom toxicity.

The present disclosure also provides a diagnostic kit for identifying (identification) the type and severity of a bite in a toxic animal. The kit contains a plurality of peptides and a plurality of marker molecules. Each peptide targets a sequence unique to the venom of one or more animal species. Each marker molecule is conjugated to a corresponding unique peptide. Blood from the victim is drawn and contacted with the peptide/tag conjugate. The kit further comprises an assay configured to detect the marker molecules, thereby revealing peptides bound to respective targets in the blood. Thus, the kit can detect which peptides bind to the target, and the extent of binding, thereby confirming the type of venom of the animal species found in the blood and the severity of the bite.

In some embodiments, disclosed herein is a multi-species antitoxin composition comprising a phage-expressed peptide that binds to a target common to more than one animal's venom; and a pharmaceutically acceptable carrier. In some embodiments, the phage-expressed peptide is from about 7 to about 12 amino acids in length; wherein the target is a metal, a carbohydrate, a protein, or any combination thereof. In some embodiments, the phage-expressed peptide is linear. In some embodiments, the phage-expressed peptide is cyclic.

In some embodiments, the target of the phage-expressed peptide is a protein. In some embodiments, the target of the phage-expressed peptide is phospholipase a₂(PLA₂)。

In some embodiments, the composition comprises one or more peptides selected from the group consisting of: 1, 2,3, 4 and any combination thereof. In some embodiments, the composition comprises a peptide comprising SEQ ID No. 1. In some embodiments, the composition comprises a peptide consisting of SEQ ID NO: 1. In some embodiments, the composition comprises a peptide comprising SEQ ID NO 2. In some embodiments, the composition comprises a peptide consisting of SEQ ID NO 2. In some embodiments, the composition comprises a peptide comprising SEQ ID No. 3. In some embodiments, the composition comprises a peptide consisting of SEQ ID NO 3. In some embodiments, the composition comprises a peptide comprising SEQ ID No. 4. In some embodiments, the composition comprises a peptide consisting of SEQ ID NO 4.

In some embodiments, the composition comprises one or more peptides selected from the group consisting of: 9, 10, 11, 12 and any combination thereof. In some embodiments, the composition comprises a peptide comprising SEQ ID No. 9. In some embodiments, the composition comprises a peptide consisting of SEQ ID NO 9. In some embodiments, the composition comprises a peptide comprising SEQ ID NO 10. In some embodiments, the composition comprises a peptide consisting of SEQ ID NO 10. In some embodiments, the composition comprises a peptide comprising SEQ ID NO 11. In some embodiments, the composition comprises a peptide consisting of SEQ ID NO 11. In some embodiments, the composition comprises a peptide comprising SEQ ID NO 12. In some embodiments, the composition comprises a peptide consisting of SEQ ID NO 12.

In some embodiments, disclosed herein are compositions comprising a phage-expressed peptide, wherein the phage-expressed peptide is expressed by a M13 phage.

In some embodiments, disclosed herein are compositions for neutralizing a plurality of animal venom, wherein the plurality of animal venom is derived from one or more snake species.

In some embodiments, disclosed herein are one or more phage-expressed peptides that bind to a target in one or more animal venom. In some embodiments, the phage-expressed peptide is selected from the group consisting of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, and any combination thereof. In some embodiments, the phage-expressing peptide targets phospholipase a₂. In still some embodiments, the one or more bacteriophage-expressed peptides include a peptide selected from the group consisting of SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, and any combination thereof. In some embodiments, the phage-expressed peptide is expressed by a M13 phage.

Also provided herein are one or more isolated nucleic acid molecules encoding any of the peptides disclosed herein. In some embodiments, the nucleic acid molecule comprises a sequence selected from the group consisting of: 5, 6,7, 8, 13, 14, 15, 16 and any combination thereof.

In some embodiments, provided herein are one or more vectors (vectors) comprising a nucleic acid encoding any one of the peptides disclosed herein. In some embodiments, the one or more vectors comprise a nucleic acid disclosed herein.

In some embodiments, provided herein are host cells comprising an isolated nucleic acid molecule disclosed herein, or an isolated vector disclosed herein. In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the host cell is e. In some embodiments, the host cell is e.coli K12ER 2738.

In some embodiments, also provided herein is a method of producing a composition disclosed herein or a peptide disclosed herein, the method comprising culturing a host cell disclosed herein under conditions in which the peptide is produced. In some embodimentsIn another aspect, provided herein is a method of producing a composition disclosed herein or a peptide disclosed herein, the method comprising (a) identifying common PLA in western cottontopodium venom₂A protein sequence; (b) identification of consensus PLA in other snake species₂A protein sequence; and (c) producing one or more phage via phage display panning.

In some embodiments, disclosed herein is a method of treating a subject in contact with animal venom, comprising administering (administerer) to the subject a composition disclosed herein. In some embodiments, the animal venom is from western cottontopodium venom. In some embodiments, the composition binds to the animal venom, thereby neutralizing venom toxicity. In some embodiments, the composition administered comprises a peptide selected from the group consisting of: 1, 2,3, 4,9, 10, 11, 12 and any combination thereof. In some embodiments, the composition is administered orally, rectally, transdermally, intravenously, intramuscularly, intraperitoneally, intramedullally, epidurally, or subcutaneously.

In some embodiments, provided herein is a diagnostic kit comprising a composition disclosed herein. In some embodiments, the kit comprises a composition comprising a peptide selected from the group consisting of: 1, 2,3, 4 and any combination thereof. In some embodiments, the kit comprises a composition comprising a peptide selected from the group consisting of: 9, 10, 11, 12 and any combination thereof.

Drawings

The following will be apparent from the elements of the drawings, which are provided for illustrative purposes and are not necessarily drawn to scale.

FIG. 1A shows PLA isolated from venom of Western Agkistrodon acutus (Aglstrodon piscivorus leucostoma) in a space-filling mold₂The crystal structure of (1). Residues have been identified using circles representing the catalytic network and the metal binding amino acids, respectively. Secondary image (secondary image)e) A 90 ° counterclockwise rotation of the crystal structure is shown to observe the active residue.

FIG. 1B is PLA from venom of Western Agkistrodon Halys₂The conserved sequence of (a), which is used as a target peptide for panning. The inner regions representing the active catalytic site (yellow) and the metal binding site (red) were used as template peptides for phage panning.

FIG. 2 is PLA when Western Agkistrodon Halys venom was incubated for 30 minutes with polyclonal phage mixture from the second round of panning₂Graphical representation of inhibition. The inhibition depends on the concentration of the polyclonal phage mixture.

FIG. 3 is a graph showing inhibition of PLA using phage clones isolated from a phage display library₂Bar graph of activity.

FIG. 4 is a cross-species anti-PLA showing the Ph.D. -12-7 phage against five major snake venoms in the United states₂Bar graph of activity. One of the selected phage clones, Ph.D. -12-7, showed anti-PLA against all major rattlesnake venoms in North America₂And (4) activity.

Fig. 5A to 5D show the steps of a method for developing universal antitoxicins from various phage display systems. FIG. 5A shows the sequence analysis of the venom components. FIG. 5B shows the affinity selection of phage. Figure 5C shows in vitro and in vivo efficacy testing. Figure 5D shows phage treatment of snake bite victims.

While the present disclosure is susceptible to various modifications and alternative forms, specific embodiments have been shown by way of example in the drawings and will be described in detail herein. However, it should be understood that the disclosure is not intended to be limited to the particular forms disclosed. Rather, the disclosure is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the disclosure as defined by the following appended claims.

Detailed Description

Provided herein are peptides and compositions comprising peptides. In some embodiments, the peptide binds snake venom and neutralizes venom toxicity. In some embodiments, the peptide binds to PLA₂And neutralize venom toxicity. In a specific embodiment, the isolation is disclosed hereinThe protein of (1).

Isolated nucleic acids (polynucleotides), such as complementary DNA (cDNA), encoding such proteins are also provided. Vectors (e.g., expression vectors) and cells (e.g., host cells) comprising nucleic acids (polynucleotides) encoding such proteins are also provided. Methods of making such proteins are also provided. In other aspects, provided herein are methods and uses for detecting snake venom. In other aspects, provided herein are methods of treating certain conditions (e.g., snake bites). Related compositions (e.g., pharmaceutical compositions), kits, and detection methods are also provided.

For the purposes of promoting an understanding of the principles of the disclosure, reference will now be made to a number of illustrative embodiments shown in the drawings and specific language will be used to describe the same.

a. Term(s) for

It should be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "a nucleotide sequence" is understood to mean one or more nucleotide sequences. Thus, the terms "a", "an", "one or more" and "at least one" are used interchangeably herein.

Further, as used herein, "and/or" will be considered to specifically disclose each of the two specified features or components, with or without the other. Thus, use of the term "and/or" in phrases such as "a and/or B" is intended to include "a and B," "a or B," "a" (alone), and "B" (alone). Likewise, the term "and/or" as used in phrases such as "A, B and/or C" is intended to encompass each of the following: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; a (alone); b (alone); and C (alone).

Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. It is also understood that all base sizes or amino acid sizes, as well as all molecular weight or molecular mass values given for a nucleic acid or polypeptide are approximate and provided for description.

It should be understood that whatever aspects are described herein by the term "comprising," other similar aspects are also provided that are described as "consisting of and/or" consisting essentially of.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, circumcise Dictionary of biomedicine and Molecular Biology, Juo, Pei-Show, 2 nd edition, 2002, CRC Press; the Dictionary of cell and Molecular Biology, 3 rd edition, 1999, Academic Press; and the Oxford dictionary Of Biochemistry And Molecular Biology, revision 2000, Oxford university Press provides one skilled in the art with a general dictionary Of many terms used in this disclosure.

Units, prefixes, and symbols are expressed in their accepted form by the Systeme International de units (SI). Numerical ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written from left to right in the amino to carboxyl orientation. The headings provided herein are not limitations of the various aspects of the disclosure which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below may be more fully defined by reference to the entire specification.

The term "about" is used herein to mean about, approximately, or within a region thereof. When the term "about" is used in conjunction with a range of values, it modifies the range by extending the boundaries above and below the stated values. Thus, "about 10-20" means "about 10 to about 20". In general, the term "about" may modify a numerical value by, for example, a 10%, upward or downward (higher or lower) magnitude of change (variance) within a range above and below the numerical value.

The term "naturally occurring" as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by man in the laboratory is naturally occurring.

"phage" or "bacteriophage" refers to a virus that infects a bacterium. The term "bacteriophage" is used to refer to both types of viruses, but in some cases, as indicated by context, may also be used as a shorthand for specifically referring to bacteriophage. Bacteriophage are obligate intracellular parasites that multiply within bacteria by exploiting some or all of the host biosynthetic machinery (i.e., the virus that infects the bacteria). Although different bacteriophages may contain different substances, they all contain nucleic acids and proteins, and in some cases may be encapsulated in a lipid membrane. Depending on the phage, the nucleic acid can be either DNA or RNA but not both, and it can exist in various forms. There are two ways in which bacteriophages can infect bacterial cells. One is lysogenicity, in which phage DNA is incorporated into the chromosome of the bacterium and becomes dormant for many generations. At least one environmental inducer is required to allow the phage DNA to be excised from the bacterial chromosome and establish the second type of infection, the lytic phase. At this stage, the bacteria are converted into a phage manufacturing plant. Hundreds of phages are generated and bacterial cells are lysed to release them. The released phage then finds another host bacterium and the process is repeated.

"M13 phage", "M13 bacteriophage" and the like are phages infected with the M13 virus. In some embodiments disclosed herein, the M13 bacteriophage is e.coli infected with M13 virus. The M13 phage consists of a circular single-stranded DNA molecule surrounded by a coating protein. In some embodiments disclosed herein, the M13 bacteriophage produces an antitoxin peptide comprising SEQ ID NOs 1-4.

An "antitoxin" is a serum that acts against venom. Antitoxic liquid is used for treating some poisonous bites and stings. In a particular embodiment herein, the antitoxic liquid is used to treat snake bites. The particular antitoxic agent required depends on the species involved. "Universal antitoxic hormones" react with the venom or venom proteins of more than one species. In other words, a universal antitoxin is an antitoxin that cross-reacts with venom of different species.

"phage display panning" is a technique that uses bacteriophage to examine protein-protein, protein-peptide, and protein-DNA interactions. Phage display panning allows enrichment of relevant phages.

"phospholipase A₂'or' PLA₂"is an enzyme belonging to the class of enzymes that hydrolyze sn-2 esters of glycerophospholipids to produce fatty acids and lysophospholipids. PLA (polylactic acid)₂Catalyzes the calcium-dependent hydrolysis of the 2-acyl group in 3-sn-phosphoglyceride, which releases glycerophospholipids and arachidonic acid as precursors to signal molecules. PLA of snake venom₂Including a very large superfamily of enzymes, which consists of 16 recognized groups of six major classes. These major classes comprise secreted PLA₂s(sPLA₂) Cytosolic PLA₂s(cPLA₂) Calcium independent PLA₂s(iPLA₂) Platelet Activating Factor (PAF) acetylhydrolase/oxidized lipid lipoprotein associated PLA₂(LpPLA₂s) fatty PLA₂s(AdPLA₂s) and lysosomal PLA₂s(LPLA₂s)。PLA₂Hydrolysis of glycerophospholipids by s results in the release of fatty acids and the associated production of lysophospholipids.

A "consensus sequence" is a sequence of nucleotides or amino acids that is shared between homologous regions in different but related DNA or RNA or protein sequences.

"polypeptide" refers to a chain comprising at least two amino acid residues joined in series, wherein the length of the chain has no upper limit. One or more amino acid residues in a protein may contain modifications such as, but not limited to, glycosylation, phosphorylation, or disulfide bond formation. A "protein" may include one or more polypeptides.

As described above, polypeptide variants comprise, for example, modified polypeptides. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamic acid, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation (Mei et al, Blood 116:270-79(2010), incorporated herein by reference in its entirety), proteolytic processing, phosphorylation, prenylation, racemization, selenization, sulfation, transfer of amino acids to proteins-RNA mediated addition (e.g., arginylation), and ubiquitination.

As used herein, "corresponding amino acids," "corresponding sites," or "equivalent amino acids" in a protein sequence are identified by alignment to maximize the identity or similarity between a first protein sequence (e.g., an IL-2 sequence) and a second protein sequence (e.g., a second IL-2 sequence). The numbers used to identify equivalent amino acids in the second protein sequence are based on the number of corresponding amino acids used to identify the first protein sequence.

The term "expression" as used herein refers to the process by which a polynucleotide produces a gene product (e.g., an RNA or polypeptide).

"conservative amino acid substitution" refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families contain amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). In some embodiments, a predicted nonessential amino acid residue in an IL-2-CD25 fusion protein is replaced with another amino acid residue from the same side chain family. Methods for confirming conservative substitution of nucleotides and amino acids that do not eliminate antigen binding are well known in the art (see, e.g., Brummell et al, biochem.32:1180-1187 (1993); Kobayashi et al Protein Eng.12(10):879-884 (1999); and Burks et al Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

The term "substantial homology," with respect to polypeptides, means that two polypeptides, or designated sequences thereof, are identical when optimally aligned and compared, with appropriate amino acid insertions or deletions in at least about 80% of the amino acids, at least about 90% to 95% of the amino acids, or at least about 98% to 99.5% of the amino acids.

"binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., a peptide disclosed herein) and its binding partner (e.g., an antigen). As used herein, unless otherwise specified, "binding affinity" refers to intrinsic binding affinity that reflects a 1: 1 interaction. The affinity of a molecule X for its partner Y can generally be determined by the dissociation constant (K)_D) And (4) showing. Affinity can be measured and/or expressed in a variety of ways known in the art, including but not limited to equilibrium dissociation constant (K)_D) And equilibrium association constant (K)_A)。K_DFrom k to k_off/k_onIs calculated by the quotient of K_AFrom k to k_on/k_offThe quotient of (2). k is a radical of_onRefers to, for example, the association rate constant of the peptide with the antigen, and k_offRefers to, for example, dissociation of the peptide from the antigen. k is a radical of_onAnd k_offCan be prepared by techniques known to those of ordinary skill in the art (e.g., by

Or KinExA).

As used herein, a "conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is substituted with an amino acid residue having a similar side chain. Families of amino acid residues having side chains have been defined in the art. These families contain amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). In certain embodiments, one or more amino acid residues within the peptides disclosed herein may be replaced with an amino acid residue having a similar side chain.

As used herein, "epitope" is a term in the art that refers to a localized region of an antigen to which a peptide can specifically bind. An epitope can be, for example, contiguous amino acids of a polypeptide [ linear or contiguous epitopes ] or an epitope can be, for example, two or more non-contiguous regions (conformational, non-linear, discontinuous, or non-contiguous epitopes) that come together from one or more polypeptides. In certain embodiments, the epitope to which a peptide disclosed herein binds can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallographic studies, ELISA assays, hydrogen/deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assays, and/or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be accomplished using any method known in the art (e.g., Gieger et al (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayenne (1997) Structure 5: 1269-.

As used herein, the term "nucleic acid molecule" is intended to encompass DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, and may be a cDNA.

The term "downstream" refers to a nucleotide sequence that is 3' to a reference nucleotide sequence. "downstream" may also refer to a peptide sequence located at the C-terminus of the reference peptide sequence.

The term "upstream" refers to a nucleotide sequence that is 5' to a reference nucleotide sequence. "upstream" may also refer to a peptide sequence that is located at the N-terminus of a reference peptide sequence.

The term "substantial homology," with respect to nucleic acids, means that two nucleic acids, or designated sequences thereof, are identical when optimally aligned and compared, with appropriate nucleic acid insertions or deletions in at least about 80% of the nucleotides, at about 90% to 95% of the nucleotides, or at least about 98% to 99.5% of the nucleotides. Alternatively, substantial homology exists when the segment (segment) hybridizes under selective hybridization conditions to the complement (complement) of the strand.

As used herein, the term "regulatory region" refers to a nucleotide sequence located upstream (5 'non-coding sequence), within, or downstream (3' non-coding sequence) of a coding region that affects transcription, RNA processing, stability, or translation of the associated coding region. Regulatory regions may comprise promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, and stem-loop structures. If the coding region is to be used for expression in eukaryotic cells, the polyadenylation signal and transcription termination sequence will generally be located 3' to the coding sequence.

A polynucleotide encoding a gene product (e.g., a polypeptide) can comprise a promoter and/or other transcriptional or translational control elements operably associated with one or more coding regions. Other transcriptional control elements besides promoters, such as enhancers, operators, repressors, and transcriptional termination signals, may also be operably associated with the coding region to direct expression of the gene product.

Various transcriptional control regions are known to those skilled in the art. These transcriptional control regions include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegalovirus (immediate early promoter, binding intron-a), simian virus 40 (early promoter), and retroviruses (e.g., rous sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes, such as actin, heat shock proteins, bovine growth hormone, and rabbit β -globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcriptional control regions include tissue-specific promoters and enhancers and lymphokine-inducible promoters (e.g., promoters induced by interferons or interleukins).

Similarly, various translational control elements are known to those of ordinary skill in the art. These translation control elements include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly internal ribosome entry sites, or IRES, also known as CITE sequences).

The terms "percent sequence identity", "percent identity", "sequence identity" or "identity" are used interchangeably and refer to the number of identical matching positions shared between two polynucleotide or polypeptide sequences over a comparison window, taking into account the additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matching position is any position that presents the same nucleotide or amino acid in both the target and reference sequences. Since gaps are not nucleotides or amino acids, gaps present in the target sequence are not counted. Likewise, gaps present in the reference sequence are not counted because nucleotides or amino acids from the target sequence are not counted as those from the reference sequence.

Sequence comparisons between two sequences and determination of percent identity can be accomplished using mathematical algorithms, as described in the non-limiting examples below.

The percentage of sequence identity is calculated by: determining the number of positions at which the same amino acid residue or nucleic acid base occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to obtain the percentage of sequence identity. Sequence comparison and determination of percent sequence identity between two sequences can be accomplished by using software online and downloading it for easy access. Suitable software programs are available from a variety of sources and are used in the alignment of both protein and nucleotide sequences. One suitable program for determining percent sequence identity is bl2seq, which is part of the BLAST program suite available from the national center for biotechnology information BLAST website of the united states government (BLAST. The Bl2seq performs a comparison between two sequences using the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water or mather, which are part of the EMBOSS bioinformatics program suite and are also available from the European Bioinformatics Institute (EBI) www.ebi.ac.uk/Tools/psa.

Different regions within a single polynucleotide or polypeptide target sequence aligned with a polynucleotide or polypeptide reference sequence may each have their own percentage of sequence identity. It should be noted that the percentage sequence identity values are rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. It should also be noted that the length value will always be an integer.

The percentage of identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available from world wide web. GCG. com) using the nwsgapdna. cmp matrix and GAP weights (weights) 40, 50, 60, 70 or 80 and length weights 1, 2,3, 4,5 or 6. The percentage identity between two nucleotide or amino acid sequences can also be determined using algorithms of e.meyers and w.miller (cabaos, 4:11-17(1989)) incorporated into the ALIGN program (version 2.0), using a PAM120 residue weight table (weight table), a gap length compensation value (penalty)12, and a gap compensation value of 4. Furthermore, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J.mol.biol. (48):444-453(1970)) algorithms of the GAP program incorporated into the GCG software package (available from http:// www.gcg.com), using either the Blossum 62 matrix or the PAM250 matrix, and GAP weights 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2,3, 4,5, or 6.

The nucleic acid and protein sequences described herein may further be used as "query sequences" to search public databases, for example, to identify related sequences. Such a search can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al (1990) J.mol.biol.215: 403-10. BLAST nucleotide searches can be performed using NBLAST programs with a score of 100 and a word length of 12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches may be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecules described herein. In order to obtain Gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic acids sRs.25 (17): 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used. See worldwidediweb. ncbi. nlm. nih. gov.

The nucleic acid may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. Nucleic acids are "isolated" or "substantially purified" when purified from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other parts of the chromosome) or proteins, by standard techniques [ including alkaline/SDS treatment, CsCl banding (banding), column chromatography, agarose gel electrophoresis, and other methods well known in the art ]. See, e.g., Ausubel, et al, ed.Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

Nucleic acids (e.g., cdnas) can be mutated according to standard techniques to provide gene sequences. For coding sequences, these mutations can affect the amino acid sequence as desired. In particular, DNA sequences substantially homologous to or derived from native V, D, J, constant sequences, switching sequences, and other such sequences described herein are contemplated (wherein "derived" indicates that a sequence is identical to or modified from another sequence).

The term "vector" as used herein means a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop to which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" are used interchangeably, as plasmids are the most commonly used form of vector. However, other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), are also encompassed and serve the same function.

As used herein, the term "recombinant host cell" (or simply "host cell") means a cell that includes a nucleic acid that does not naturally occur in the cell, and may be a cell into which a recombinant expression vector has been introduced. It is understood that these terms refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Exemplary host cells include, but are not limited to, prokaryotic cells (e.g., E.coli), or alternatively, eukaryotic cells, such as fungal cells (e.g., yeast cells, such as Saccharomyces cerevisiae, Pichia pastoris, or Schizosaccharomyces pombe), as well as various animal cells, such as insect cells (e.g., Sf-9) or mammalian cells (e.g., HEK293F, CHO, COS-7, NIH-3T 3).

The phrase "immediately downstream of an amino acid" as used herein refers to a position immediately adjacent to the carboxyl group at the end of the amino acid. Similarly, the phrase "immediately upstream of an amino acid" refers to a position immediately adjacent to the terminal amine group of the amino acid. Thus, the phrase "between two amino acids of an insertion site" as used herein refers to a location where a heterologous moiety (e.g., a half-life extending moiety) is inserted between two adjacent amino acids.

"treating" or "treatment" as used herein refers to, for example, a reduction in the severity of a disease or disorder; shortening the duration of the disease course; amelioration or elimination of one or more symptoms associated with the disease or disorder; providing a beneficial effect to a subject suffering from a disease or condition without having to cure the disease or condition.

As used herein, "administering" refers to physically introducing a composition comprising a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Different routes of administration of the peptides described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral (e.g., by injection or infusion) routes of administration. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, typically by injection, including but not limited to intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcontracting, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. Alternatively, the peptides described herein may be administered via a non-parenteral route, for example via a topical, epidermal or mucosal route, for example via an intranasal, oral, vaginal, rectal, sublingual or topical route. Administration may also be performed, for example, once, multiple times, and/or over one or more extended periods of time.

By "vaccine" is meant a composition for stimulating a specific immune response (or immunogenic response) in a subject. In some embodiments, the immunogenic response is protective or provides protective immunity. For example, in the case of a pathogenic organism, a vaccine enables a subject to better resist infection by the organism against which the vaccine is directed or to resist disease exacerbations caused by that organism. Alternatively, in the case of cancer, the vaccine boosts the subject's natural defense against already-exacerbated cancer. These types of vaccines may also prevent further progression of current cancers, prevent recurrence of the treated cancer and/or eliminate cancer cells that were not killed by previous treatments.

As used herein, the term "effective amount" in the context of administering a therapy to a subject refers to the amount of the therapy that achieves the desired prophylactic or therapeutic effect.

As used herein, the terms "subject" and "patient" are used interchangeably. The subject may be an animal. In some embodiments, the subject is a mammal, e.g., a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkey or human), most preferably a human. In certain embodiments, such terms refer to a non-human animal (e.g., a non-human animal such as a pig, horse, cow, cat, or dog). In some embodiments, such terms refer to a pet or farm animal. In a specific embodiment, such term refers to a human.

As used herein, the terms "ug" and "uM" are used interchangeably with "μ g" and "μ Μ", respectively.

Various aspects described herein are described in more detail in the following subsections.

b. Peptides

The disclosure also identifies phage-expressed peptides that strongly bind to conserved snake venom components and neutralize venom toxicity. In some embodiments, disclosed herein are peptides that strongly bind to a conserved snake venom component. In some embodiments, disclosed herein are peptides that neutralize venom toxicity. In some embodiments, disclosed herein are peptides that strongly bind to conserved snake venom components and neutralize venom toxicity.

Unlike, for example, antibody-based anti-venom therapies limited to antigen targeting, phage display of the present disclosure provides a powerful tool for selecting phage-expressed peptides that bind many different targets with high specificity and affinity. These phage-expressed peptides are short, usually 7-12 mer fragments, whose structure can be linear or cyclic. Examples include cyclic 7-mer and linear 12-mer peptides.

In one embodiment, disclosed herein is a linear 7-mer. In one embodiment, disclosed herein is a linear 8 mer. In one embodiment, disclosed herein is a linear 9 mer. In one embodiment, disclosed herein is a linear 10 mer. In one embodiment, disclosed herein is a linear 11 mer. In one embodiment, disclosed herein is a linear 12 mer.

In one embodiment, disclosed herein is a cyclic 7-mer. In one embodiment, disclosed herein are cyclic 8-mers. In one embodiment, disclosed herein are cyclic 9-mers. In one embodiment, disclosed herein are cyclic 10-mers. In one embodiment, disclosed herein is a cyclic 11-mer. In one embodiment, disclosed herein are cyclic 12-mers.

Metals, carbohydrates and proteins are the major components of animal venom. The peptides disclosed herein can target a number of macromolecules. In some embodiments, disclosed herein are peptides that target metals, carbohydrates, and proteins. In some embodiments, disclosed herein are peptides that target metals and carbohydrates. In some embodiments, disclosed herein are peptides that target metals and proteins. In some embodiments, disclosed herein are peptides that target carbohydrates and proteins. In some embodiments, the peptides disclosed herein target carbohydrates. In some embodiments, disclosed herein are peptides that bind to proteins. In one embodiment, the peptides disclosed herein target metals.

Disclosed herein are peptides comprising a sequence selected from SEQ ID NOs 1-4. In one embodiment, disclosed herein is a peptide (SPLHKTM; also referred to as Ph.D. -C7C-6) comprising SEQ ID NO: 1. In one embodiment, disclosed herein is a peptide comprising SEQ ID NO:2 (SGMKKTK; also known as Ph.D. -C7C-7). In one embodiment, disclosed herein is a peptide comprising SEQ ID NO:3 (KTTKMGL; also known as Ph.D. -C7C-9). In one embodiment, disclosed herein is a peptide comprising SEQ ID NO:4 (KLIHGNGVMDEG; also referred to as Ph.D. -12-2 or Ph.D. -12-7).

In some embodiments, disclosed herein are biologically active variants of SEQ ID NOs 1-4. In some embodiments, biologically active fragments and variants of SEQ ID NOs 1-4 comprise at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to the sequences set forth in SEQ ID NOs 1-4.

In some embodiments, the biologically active variant comprises one or more additional amino acids compared to SEQ ID NOs 1-4. In some embodiments, the biologically active variant comprises one or fewer amino acids as compared to SEQ ID NOS: 1-4.

In some embodiments, a biological variant of a peptide disclosed herein comprises one or more mutations. In some embodiments, the mutation is a substitution mutation. In some embodiments, the substitution is a conservative substitution of amino acids that do not affect protein folding and/or activation. Examples of conservative substitutions belong to the group consisting of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and aspartic acid), hydrophobic amino acids (leucine, isoleucine, valine and methionine), aromatic amino acids (phenylalanine, tryptophan and tyrosine) and small amino acids (glycine, alanine, serine and threonine). Amino acid substitutions which do not generally alter specific activity are known in the art of the present invention. The most commonly occurring alterations are Ala/Ser, Vai/IIe, Asp/Giu, Thr/Ser, Ala/Giy, Ala/Thr, Ser/Asn, Ala/Val, Ser/Giy, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/IIe, Leu/Val, Ala/Giu, Asp/Giy and vice versa.

In some embodiments, disclosed herein are reverse consensus peptide sequences of SEQ ID NOs 1-4. In one embodiment, the reverse consensus peptide sequence comprises SEQ ID NO 9. In one embodiment, the reverse consensus peptide sequence comprises SEQ ID NO 10. In one embodiment, the reverse consensus peptide sequence comprises SEQ ID NO 11. In one embodiment, the reverse consensus peptide sequence comprises SEQ ID NO 12.

In another embodiment of the present disclosure, peptides having the reverse consensus sequence of SEQ ID NO 1-4 are used to develop a diagnostic kit for bites or stings. The kits are described in more detail below.

Table 1:

most antibody-based solutions either require special storage conditions or, in the case of lyophilization, require reconstitution prior to administration; both of which reduce their utility in remote and severe conditions where animal toxicity often occurs. Disclosed herein are peptide solutions that do not require special storage conditions.

In some embodiments, the peptides disclosed herein are stable at room temperature. In some embodiments, the peptides disclosed herein are stable at-80 ℃. In some embodiments, the peptides disclosed herein are stable at-80 ℃. In some embodiments, the peptides disclosed herein are stable at-70 ℃. In some embodiments, the peptides disclosed herein are stable at-60 ℃. In some embodiments, the peptides disclosed herein are stable at-50 ℃. In some embodiments, the peptides disclosed herein are stable at-40 ℃. In some embodiments, the peptides disclosed herein are stable at-30 ℃. In some embodiments, the peptides disclosed herein are stable at-20 ℃. In some embodiments, the peptides disclosed herein are stable at-10 ℃. In some embodiments, the peptides disclosed herein are stable at 0 ℃. In some embodiments, the peptides disclosed herein are stable at 10 ℃. In some embodiments, the peptides disclosed herein are stable at 20 ℃. In some embodiments, the peptides disclosed herein are stable at 30 ℃. In some embodiments, the peptides disclosed herein are stable at 40 ℃. In some embodiments, the peptides disclosed herein are stable at 50 ℃.

In one embodiment, the peptides disclosed herein include PLA of western cottontopodium₂A consensus peptide. In a particular embodiment, venom from a p.leucostoma snake is passed through ph.d.^TM-7、Ph.D.^TM-12 and ph.d^TMC7C phage display peptide library (New England)

Inc., Ipswich, MA) and screened. PLA was selected to inhibit 35% to 60%, as described below₂Four unique monoclonal anti-PLA's of activity₂Phage clones.

c. Peptide production

The M13 bacteriophage of one embodiment of the present disclosure can be inexpensively and safely propagated in specialized e.coli K12ER2738 lacking common pathogenicity-related sequences. The resulting product can be purified more efficiently than antisera.

The development of a particular antitoxic formulation using the methods of the present disclosure is fast and simple enough to allow customization to meet specific needs. One selection cycle will take one week and the entire selection of candidate phage can be completed in about 2 to 6 months. Because the synthesis of phage-based antidotes according to the present disclosure can utilize non-pathogenic strains of E.coli without the need for large or dangerous animals, the risk and burden of maintaining such animals in a laboratory environment is avoided.

Common PLA based on western cottony pallas venom₂Peptide target design of protein sequences thus provides a useful target for affinity assignment of phage display libraries. Test and venom component (PLA) Using ELISA₂Or protease) activity assay can be used to assess the effectiveness of a display phage-based antitoxin. The silica approach of epitope targeting (in silica approach) allows for universal targeting of protein families or superfamilies, e.g., targeting PLA in a specific genus₂。PLA₂The active region has-95% homology in species of agkistrodon halys (a. piscovorus). Random targeting of the target sequence results in several binders expressing variable motifs that inhibit activity by different mechanisms, resulting in a stronger inhibitory effect. When extended to vipers common in North America, PLA is now present₂The homology of the construct was reduced to 56% in the western rattlesnake (Crotalus atrox). Redefining the target sequence to include homologous regions of the five most common vipers in north america can increase the consensus sequence to 97%. The target peptides are further defined to mimic conserved activities and metal binding sites in these homologous regions of the peptide family, thereby providing an improved method for developing universal antitoxicins. Their structural and functional relationships are generally known. However, this knowledge has not prompted the development of universal antitoxic agents due to the limitations of antibody-based antitoxic fluid production methods. However, the phage display method of the present disclosure can be implemented using the results of similarity search using the BlastX algorithm. The venom sequences can then be classified and used as target peptides to develop phage-based antivirals. Thus, universal antitoxicins can be developed that target the consensus sequence of any reptile, spider, jellyfish, or other toxic animal.

In some embodiments, disclosed herein are methods of producing the peptides disclosed herein. The method comprises the steps ofCommon PLA in Western cottony Agkistrodon venom₂Protein sequence validation peptide targets to provide targets for phage display panning; redefining the target sequence to comprise homologous regions of at least five common vipers in north america; and redefining the target peptide by targeting a conserved active site to mimic the homologous region of the peptide family.

Exemplary North American snakes include, but are not limited to, eastern Trapa maculans (Crotalus amantanus), Western Trapa maculans (Crotalus atrox), Western coral snakes (Micruroideusuryx), eastern coral snakes (Micrurus fulvius), Brachydon acutus (Agkistrodon contortrix), Agkistrodon halys pallas (Agkistrodon contortrix), yellow belly sea snakes (Pelamis platura), woodgrain rattlesnake (Crotalus hordus), Mohara rattlesnake (Crotalus scutus), and black rattlesnake (Sichurus cathatus).

d. Nucleotide, its preparation and use

In certain aspects, provided herein are polynucleotides comprising nucleotide sequences encoding the peptides described herein that bind to conserved snake venom components and neutralize venom toxicity. In some embodiments, polynucleotides are disclosed herein.

Disclosed herein are polynucleotides comprising a sequence selected from SEQ ID NOs 5-8. In one embodiment, disclosed herein is a polynucleotide comprising SEQ ID NO 5. In one embodiment, disclosed herein is a polynucleotide comprising SEQ ID NO 6. In one embodiment, disclosed herein is a polynucleotide comprising SEQ ID NO. 7. In one embodiment, disclosed herein is a polynucleotide comprising SEQ ID NO 8.

Also disclosed herein are polynucleotides comprising a sequence selected from SEQ ID NOS 13-16. In one embodiment, disclosed herein is a polynucleotide comprising SEQ ID NO 13. In one embodiment, disclosed herein is a polynucleotide comprising SEQ ID NO. 14. In one embodiment, disclosed herein is a polynucleotide comprising SEQ ID NO. 15. In one embodiment, disclosed herein are polynucleotides comprising SEQ ID NO 16

Provided herein are polynucleotides comprising nucleotide sequences encoding any of the peptides provided herein, as well as vectors comprising such polynucleotide sequences, e.g., expression vectors for the efficient expression thereof in a host cell, e.g., M13 bacteriophage.

As used herein, an "isolated" polynucleotide or nucleic acid molecule is a polynucleotide or nucleic acid molecule that is separate from other nucleic acid molecules present in the natural source of the nucleic acid molecule (e.g., mouse or human). In addition, an "isolated" nucleic acid molecule, such as a cDNA molecule, is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. For example, the term "substantially free" of a preparation (preparation) comprising a polynucleotide or nucleic acid molecule has less than about 15%, 10%, 5%, 2%, 1%, 0.5% or 0.1% (especially less than about 10%) of other materials, such as cellular material, culture medium, other nucleic acid molecules, chemical precursors and/or other chemicals. In a specific embodiment, a nucleic acid molecule encoding a peptide described herein is isolated or purified.

Also provided herein are polynucleotides encoding peptides comprising SEQ ID NOS: 1-4 and SEQ ID NOS: 13-16, optimized, for example, by codon/RNA optimization, replacement with a heterologous signal sequence, and elimination of mRNA instability elements. Methods of generating optimized nucleic acids encoding the peptides disclosed herein for recombinant expression by introducing codon changes and/or elimination of the suppression region in mRNA can be achieved by adjusting, for example, U.S. patent No. 5,965,726; 6,174,666, respectively; 6,291,664, respectively; 6,414,132, respectively; and 6,794,498, respectively. For example, potential splice sites and labile elements (e.g., A/T or A/U rich elements) within an RNA can be mutated without altering the amino acids encoded by the nucleic acid sequence to increase the stability of the RNA for recombinant expression. These alterations take advantage of the degeneracy of the genetic code, e.g., the use of alternative codons for the same amino acid. In some embodiments, it may be desirable to alter one or more codons to encode conservative mutations, e.g., similar amino acids having similar chemical structures and properties and/or functions as the original amino acid. Such methods can result in an increase in expression of a peptide disclosed herein by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more relative to expression of a peptide disclosed herein encoded by an unoptimized polynucleotide.

In certain embodiments, an optimized polynucleotide sequence encoding a peptide disclosed herein can hybridize to an antisense (e.g., complementary) polynucleotide encoding an unoptimized polynucleotide sequence of a peptide described herein.

The polynucleotide may be obtained by any method known in the art and the nucleotide sequence of the polynucleotide determined. The nucleotide sequence encoding a peptide described herein can be determined using methods well known in the art, i.e., the nucleotide codons known to encode a particular amino acid are assembled in a manner that generates a nucleic acid encoding the peptide. Such polynucleotides encoding the peptides disclosed herein can be assembled from chemically synthesized oligonucleotides [ e.g., as described in Kutmeier G et al, (1994), BioTechniques 17:242-6) ], which briefly involves synthesis of overlapping oligonucleotides containing portions of the sequence encoding the peptide, annealing and ligation of those oligonucleotides, followed by amplification of the ligated oligonucleotides by PCR.

Alternatively, polynucleotides encoding the peptides described herein can be generated from nucleic acids of suitable sources (e.g., hybridomas) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers that hybridize to the 3 'and 5' ends of known sequences can be performed using genomic DNA obtained from hybridoma cells that produce the peptide of interest. The amplified nucleic acid can be cloned into a vector for expression in a host cell and further cloning.

Nucleic acids encoding the peptides disclosed herein can be chemically synthesized or obtained from suitable sources by PCR amplification using synthetic primers that hybridize to the 3 'and 5' ends of the sequences or by cloning using oligonucleotide probes specific for particular gene sequences to identify, for example, cDNA clones from a cDNA library encoding the peptides disclosed herein. The amplified nucleic acid generated by PCR may then be cloned into a replicable cloning vector using any method well known in the art.

The DNA-encoding peptides described herein can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes).

Also provided are polynucleotides that hybridize to polynucleotides encoding the peptides described herein under high, medium, or low stringency hybridization conditions. Hybridization conditions have been described in the art and are known to those skilled in the art. For example, hybridization under stringent conditions can involve hybridization to filter-bound DNA in 6 XSSC/sodium citrate (SSC) at about 45 ℃ followed by one or more washes in 0.2 XSSC/0.1% SDS at about 50-65 ℃; hybridization under highly stringent conditions can involve hybridization to filter-bound nucleic acid in 6XSSC at about 45 ℃ followed by one or more washes in 0.1 XSSC/0.2% SDS at about 68 ℃. Hybridization under other stringent hybridization conditions is known to those skilled in the art and has been described, see, e.g., Ausubel FM et al, eds., (1989) Current protocols Molecular Biology, Vol.I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York, pages 6.3.1-6.3.6 and 2.10.3.

e. Cells and vectors

In certain aspects, provided herein are expression (e.g., recombinant expression) specific binding to PLA₂And related polynucleotides, cells (e.g., host cells) of the peptides (or antigen-binding fragments thereof) described herein, and expression vectors. Provided herein are vectors (e.g., expression vectors) comprising a polynucleotide comprising a nucleotide sequence encoding a peptide or fragment disclosed herein for recombinant expression in a host cell. Also provided herein are host cells comprising such vectors for recombinant expression of the peptides described herein. In a particular aspect, provided herein are methods for producing a peptide described herein, comprising expressing such a peptide from a host cell.

In some embodiments, the M13 phage expresses a peptide disclosed herein. The M13 phage was used as a delivery vehicle to transport various binding motifs to targets. In some embodiments, genetic modification of the phage tail protein allows for expression of unique peptides with variable sequence, length, and composition. The expressed peptides can bind to specific epitopes, thus forming the basis of a high-throughput system for the identification of binding partners. In some embodiments, the M13 phage expresses a peptide selected from the group consisting of SEQ ID NOs 1-4.

In some embodiments, the M13 phage expresses a peptide comprising SEQ ID NO 1. In some embodiments, the M13 phage expresses a peptide comprising SEQ ID NO 2. In some embodiments, the M13 phage expresses a peptide comprising SEQ ID NO 3. In some embodiments, the M13 phage expresses a peptide comprising SEQ ID NO. 4.

In some embodiments, the M13 phage expresses a peptide comprising SEQ ID NO 13. In some embodiments, the M13 phage expresses a peptide comprising SEQ ID NO. 14. In some embodiments, the M13 phage expresses a peptide comprising SEQ ID NO. 15. In some embodiments, the M13 phage expresses a peptide comprising SEQ ID NO 16.

In some embodiments, the M13 phage has a long plasma half-life (t 1/2 ═ 4.5 hours).

The M13 phage disclosed herein is stable over a pH range of 3-11. In some embodiments, the pH of the M13 phage is 3. In some embodiments, the pH of the M13 phage is 4. In some embodiments, the pH of the M13 phage is 5. In some embodiments, the pH of the M13 phage is 6. In some embodiments, the pH of the M13 phage is 7. In some embodiments, the pH of the M13 phage is 7.4. In some embodiments, the pH of the M13 phage is 8. In some embodiments, the pH of the M13 phage is 9. In some embodiments, the pH of the M13 phage is 10. In some embodiments, the pH of the M13 phage is 11.

The M13 phage disclosed herein also tolerates temperatures below 80 ℃.

The M13 phage disclosed herein has a half-life of more than 6 months in culture medium at room temperature without any special storage conditions.

Once the polynucleotide encoding the peptide described herein is obtained, the vector for producing the peptide molecule can be produced by recombinant DNA techniques using techniques well known in the art.

Thus, described herein are methods for producing a protein by expressing a polynucleotide comprising a nucleotide sequence encoding the protein. Expression vectors containing the protein coding sequence and appropriate transcriptional and translational control signals can be constructed using methods well known to those skilled in the art. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Replicable vectors are also provided, which include a nucleotide sequence encoding a protein described herein operably linked to a promoter.

The expression vector can be transferred to a cell (e.g., a host cell) by conventional techniques, and the resulting cell can then be cultured by conventional techniques to produce the proteins described herein. Accordingly, provided herein are host cells containing a polynucleotide encoding a protein described herein or a fragment thereof operably linked to a promoter for expression of such sequence in the host cell.

Various host expression vector systems can be utilized to express the peptides described herein. Such host expression systems represent vehicles by which a coding sequence of interest can be produced and subsequently purified, but also represent cells that can express a peptide as described herein in situ upon transformation or transfection with an appropriate nucleotide coding sequence. These include, but are not limited to, microorganisms, such as bacteria (e.g., E.coli and Bacillus subtilis), transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing peptide coding sequences; yeast (e.g., pichia pastoris) transformed with a recombinant yeast expression vector containing a peptide coding sequence; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing peptide coding sequences; plant cell systems (e.g., green algae such as Chlamydomonas reinhardtii) infected with a recombinant viral expression vector (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with a recombinant plasmid expression vector (e.g., Ti plasmid) containing a peptide coding sequence; or mammalian cell systems [ e.g., COS (such as COS1 or COS), CHO, BHK, MDCK, HEK293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH3T3, HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB/20, and BMT10 cells ] carrying recombinant expression constructs containing promoters derived from the genome of mammalian cells (such as the metallothionein promoter) or from mammalian viruses (such as the adenovirus late promoter; the vaccinia virus 7.5K promoter).

In some embodiments, the host cell is escherichia coli. In a particular embodiment, the host cell is E.coli K12ER 2738.

In some embodiments, the cell used to express the peptides described herein is a human cell, e.g., a human cell line. In a specific embodiment, the mammalian expression vector is pOptiVEC^TMOr pcDNA3.3. In a particular embodiment, it is a bacterial cell such as E.coli or a eukaryotic cell (e.g., a mammalian cell).

In bacterial systems, a number of expression vectors can be used. For example, when large quantities of such peptides are to be produced, vectors directing the expression of high levels of protein products that are readily purified may be required in order to produce pharmaceutical compositions of the peptide molecules. Such vectors include, but are not limited to, the E.coli expression vector pUR278(Rueth U & Mueller-Hill B (1983) EMBO J2: 1791-1794) in which the peptide coding sequence may be ligated separately into a vector in frame with the lac Z coding region, thereby producing a fusion protein; pIN vector (Inouye S & Inouye M (1985) Nuc Acids Res 13: 3101-; and so on. For example, pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST).

In insect systems, for example, Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector for expressing foreign genes. The virus grows in Spodoptera frugiperda cells. The peptide coding sequence may be cloned separately into a non-essential region of the virus (e.g., the polyhedrin gene) and placed under the control of an AcNPV promoter (e.g., the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems can be utilized. In the case of using an adenovirus as an expression vector, the peptide coding sequence of interest can be linked to an adenovirus transcription/translation control complex, such as the late promoter and tripartite leader sequence. The chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into non-essential regions of the viral genome (e.g., regions E1 or E3) will result in recombinant viruses that are viable and capable of expressing peptide molecules in infected hosts (see, e.g., Logan J & Shenk T (1984) PNAS 81(12): 3655-9). Specific initiation signals may also be required for efficient translation of the inserted peptide coding sequence. These signals contain the ATG initiation codon and adjacent sequences. In addition, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of natural and synthetic origins. Expression efficiency can be enhanced by inclusion of appropriate transcription enhancer elements, transcription terminators, and the like (see, e.g., Bitter G et al, (1987) Methods enzymol.153: 516-.

In addition, host cell strains may be selected which regulate the expression of the inserted sequences or modify and process the gene product in the particular manner desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of the protein product may be important for the function of the protein. Different host cells have characteristics and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems may be selected to ensure proper modification and processing of the expressed foreign protein. For this purpose, eukaryotic host cells can be used which have cellular mechanisms for rational processing, glycosylation and phosphorylation of the primary transcript of the gene product. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK293, NIH3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (not a murine myeloma cell line endogenously producing any immunoglobulin chain), CRL7O3O, COS (such as COS1 or COS), PER. C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB/20, BMT10 and HsS78Bst cells.

In a specific embodiment, the peptides described herein have a reduced fucose content or an afucose content. Such peptides can be produced using techniques known to those skilled in the art. For example, fucose may be absent or absentThe peptide is expressed in cells with the ability to be glycosylated. In a particular example, a cell line having both alleles of a knockout α 1, 6-fucosyltransferase can be used to produce peptides having reduced fucose content.

The system (Lonza) is an example of such a system that can be used to produce peptides with reduced fucose content.

For long-term high-yield production of recombinant proteins, stable expression cells can be generated.

In certain aspects, rather than using an expression vector containing a viral origin of replication, a host cell can be transformed with DNA controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. Following the introduction of the exogenous DNA/polynucleotide, the engineered cells may be allowed to grow for 1-2 days in enriched media and then switched to selective media. Selectable markers in recombinant plasmids confer resistance to selection and allow cells to stably integrate the plasmid into their chromosomes and grow to form foci, which in turn can be cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines that express the peptides described herein. Such engineered cell lines are particularly useful for screening and evaluating compositions that interact directly or indirectly with peptide molecules.

A number of selection systems can be used, including, but not limited to, herpes simplex virus thymidine kinase (Wigler M et al, (1977) Cell 11(1):223-32), hypoxanthine guanine phosphoribosyl transferase (Szybalska EH & Szybalski W (1962) PNAS 48(12):2026-2034) and adenine phosphoribosyl transferase (Lowy I et al, (1980) Cell22(3):817-23) genes for tk cells, hgprt cells or aprt cells, respectively. Furthermore, antimetabolite resistance can be used as the basis for selection of the following genes: dhfr, which confers resistance to methotrexate (Wigler M et al, (1980) PNAS 77(6): 3567-70; O' Hare K et al, (1981) PNAS 78: 1527-31); gpt, which confers resistance to mycophenolic acid (Mulligan RC & Berg P (1981) PNAS 78(4): 2072-6); neo which confers resistance to the aminoglycoside G-418 (Wu GY & Wu CH (1991) Biotherapy 3: 87-95; Tolstoshiev P (1993) Ann Rev Pharmacol Toxicol 32: 573-596; Mulligan RC (1993) Science 260: 926-932; and Morgan RA & Anderson WF (1993) Ann Rev Biochem 62: 191-217; Nabel GJ & Felgner PL (1993) Trends Biotechnol 11(5): 211-5); and hygro, which confers resistance to hygromycin (Santerre RF et al, (1984) Gene 30(1-3): 147-56). Methods generally known in the art of recombinant DNA technology can be routinely applied to select the desired recombinant clone and are described, for example, in Ausubel FM et al, (eds.), Current Protocols in molecular Biology, John Wiley & Sons, NY (1993); kriegler M, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and Dracopoli NC et al, (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); colb re-Garapin F et al, (1981) J Mol Biol 150: chapters 12 and 13 of 1-14, the entire contents of which are incorporated herein by reference.

Expression levels of peptides can be increased by vector amplification [ see review in Bebbington CR & Hentschel CCG, The use of vector based on gene amplification for The expression of genes in mammalian cells DNA cloning, Vol.3(Academic Press, New York,1987) ]. When the marker in the vector system expressing the peptide is amplifiable, an increase in the level of inhibitor present in the host cell culture will increase the copy number of the gene marker. Since the amplified region is associated with a peptide gene, peptide production will also be increased (Crouse GF et al, (1983) Mol Cell Biol 3: 257-66).

Once the peptides described herein are produced by recombinant expression, they may be purified by any method known in the art for purifying immunoglobulin molecules, such as by chromatography (e.g., ion exchange; affinity, particularly by affinity for specific antigens following protein A; and also by column chromatography), centrifugation, differential solubilization (differential solubilization), or by any other standard technique for protein purification. In addition, the peptides described herein can be fused to heterologous polypeptide sequences described herein or other heterologous polypeptide sequences known in the art to facilitate purification.

In particular embodiments, the peptides described herein are isolated or purified. Typically, an isolated peptide is a peptide that is substantially free of other peptides having different antigenic specificities than the isolated peptide. For example, in a particular embodiment, the preparation of peptides described herein is substantially free of cellular material and/or chemical precursors. The term "substantially free of cellular material" encompasses preparations of peptides wherein the peptides are separated from cellular components of the cells from which the peptides are isolated or recombinantly produced. Thus, a peptide that is substantially free of cellular material comprises a peptide preparation having less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (by dry weight) of heterologous protein (also referred to herein as "contaminating protein") and/or variants of the peptide. When the peptide is recombinantly produced, it is also typically substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of the protein preparation. When a peptide is produced by chemical synthesis, it is typically substantially free of chemical precursors or other chemicals, i.e., it is separate from the chemical precursors or other chemicals involved in protein synthesis. Thus, such preparations of the peptide have less than about 30%, 20%, 10% or 5% (by dry weight) of chemical precursors or compounds other than the peptide of interest. In a specific embodiment, the peptides described herein are isolated or purified.

f. Composition and pharmaceutical composition

Disclosed herein are compositions comprising peptides comprising a sequence selected from SEQ ID NOs 1-4 and 9-12. Also disclosed herein are compositions comprising peptides comprising SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 12, or any combination thereof.

In one embodiment, disclosed herein is a composition comprising a peptide comprising SEQ ID NO: 1. In one embodiment, disclosed herein is a composition comprising a peptide comprising SEQ ID No. 2. In one embodiment, disclosed herein is a composition comprising a peptide comprising SEQ ID No. 3. In one embodiment, disclosed herein is a composition comprising a peptide comprising SEQ ID No. 4. In one embodiment, disclosed herein is a composition comprising a peptide comprising SEQ ID NO 9. In one embodiment, disclosed herein is a composition comprising a peptide comprising SEQ ID NO 10. In one embodiment, disclosed herein is a composition comprising a peptide comprising SEQ ID NO 11. In one embodiment, disclosed herein is a composition comprising a peptide comprising SEQ ID NO. 12.

In some embodiments, the peptides disclosed herein are formulated with a range of alternative delivery systems (e.g., nanoparticles). In some embodiments, compositions are provided that include a nanoparticle and a peptide disclosed herein. In some embodiments, the present disclosure provides an aqueous liposomal nanoparticle composition comprising an aqueous dispersion of liposomal nanoparticles and a peptide disclosed herein. In some embodiments, the nanoparticle encapsulates a peptide disclosed herein. In some embodiments, the peptides disclosed herein are added to a preformed liposome composition. In other embodiments, the peptides disclosed herein are incorporated into liposomes during formation of the liposomes.

Also provided herein are compositions comprising the peptides described herein having a desired purity in a physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1990) mack publishing co. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and comprise: buffers such as phosphate, citrate and other organic acids; an antioxidant comprising ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride, benzethonium chloride; phenol alcohol, butanol or benzyl alcohol; alkyl parabens (such as methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucoseGlucose, mannose or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and/or nonionic surfactants, e.g. TWEEN^TM、PLURONICS^TMOr polyethylene glycol (PEG).

In a specific embodiment, the pharmaceutical composition comprises a peptide described herein and optionally one or more additional prophylactic or therapeutic agents in a pharmaceutically acceptable carrier. In a specific embodiment, the pharmaceutical composition comprises an effective amount of a peptide described herein and optionally one or more additional prophylactic therapeutic agents in a pharmaceutically acceptable carrier. In some embodiments, the peptide is the only active ingredient contained in the pharmaceutical composition. The pharmaceutical compositions described herein are useful for strongly binding conserved snake venom components and neutralizing venom toxicity.

Pharmaceutically acceptable carriers for parenteral formulations include aqueous vehicles (aquous vehicles), non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, chelating or complexing agents and other pharmaceutically acceptable substances. Examples of aqueous vehicles include sodium chloride injection, ringer's injection, isotonic glucose injection, sterile water injection, dextrose and lactated ringer's injection. Non-aqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil and peanut oil. Antimicrobial agents at bacteriostatic or fungistatic concentrations may be added to parenteral formulations packaged in multi-dose containers comprising phenols or cresols, mercurial, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. The buffer comprises phosphate and citrate. The antioxidant comprises sodium bisulfate. The local anesthetic comprises procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropylmethylcellulose and polyvinylpyrrolidone. The emulsifier comprises polysorbate 80: (

80). The chelating or complexing agent for metal ions comprises EDTA. The pharmaceutical carrier also comprises ethanol, polyethylene glycol and propylene glycol for a water miscible vehicle; and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.

The pharmaceutical composition may be formulated for administration to a subject by any route. Specific examples of routes of administration include intranasal, oral, pulmonary, transdermal, intradermal, and parenteral. Parenteral administration characterized by subcutaneous, intramuscular, or intravenous injection is also contemplated herein. Injectables can be prepared in conventional forms as liquid solutions or suspensions, solid forms suitable for dissolution or suspension in liquid prior to injection, or as emulsions. Injections, solutions and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. In addition, if desired, the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers, and other such agents, such as, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrins.

Formulations for parenteral administration of compositions comprising the peptides disclosed herein comprise sterile solutions for injection; sterile drying of soluble products, such as lyophilized powders, which are combined with a solvent just prior to use, including subcutaneous injection tablets; sterile suspensions for injection; aseptically drying the insoluble product, which is combined with the vehicle just prior to use; and sterile emulsions. The solution may be an aqueous solution or a non-aqueous solution.

Suitable carriers, if administered intravenously, include physiological saline or Phosphate Buffered Saline (PBS), as well as solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol, and mixtures thereof.

The described preparation includes topical mixtures of compositions comprising the peptides disclosed herein for local and systemic administration. The resulting mixture may be a solution, suspension, emulsion, etc., and may be formulated as a cream, gel, ointment, emulsion, solution, elixir, lotion, suspension, tincture, paste, foam, aerosol, rinse, spray, suppository, bandage, skin patch, or any other formulation suitable for topical administration.

Compositions comprising the peptides disclosed herein may be formulated for topical or topical application, for example in the form of gels, creams and emulsions for topical application to the skin and mucous membranes (e.g., in the eye), as well as for application to the eye or for intracisternal or intraspinal application. Topical administration is envisaged for transdermal delivery, also for administration to the eye or mucosa, or for inhalation therapy. Nasal solutions of the peptides alone or in combination with other pharmaceutically acceptable excipients may also be administered.

Transdermal patches containing iontophoresis and electrophoresis devices are well known to those skilled in the art and can be used to administer the peptides disclosed herein. Such patches are disclosed, for example, in U.S. patent nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957, each of which is incorporated by reference in its entirety.

In certain embodiments, the pharmaceutical compositions comprising the peptides described herein are lyophilized powders that can be reconstituted for administration as solutions, emulsions, and other mixtures. It can also be reconstituted and formulated as a solid or gel. A lyophilized powder is prepared by dissolving the peptide or pharmaceutically acceptable derivative thereof described herein in a suitable solvent. In some embodiments, the lyophilized powder is sterile. The solvent may contain excipients that improve the stability or other pharmacological components of the powder or reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable agents. The solvent may also contain a buffer, such as citrate, sodium or potassium phosphate or other such buffers known to those skilled in the art, in one embodiment a buffer having a pH of about neutral. The solution is then sterile filtered and then lyophilized under standard conditions known to those skilled in the art to give the desired formulation. In one embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial will contain a single dose or multiple doses of the compound. The lyophilized powder can be stored under appropriate conditions (e.g., about 4 ℃ to room temperature).

Reconstitution of the lyophilized powder with water for injection provides a formulation for parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable carrier. The exact amount depends on the compound selected. Such amounts may be determined empirically.

The peptides described herein and other compositions provided herein can also be formulated to target specific tissues, receptors, or other regions of the body of the subject to be treated. Many such targeting methods are well known to those skilled in the art. All such targeting methods for use in the present compositions are contemplated herein. For non-limiting examples of targeting approaches, see, e.g., U.S. patent nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542, and 5,709,874, each of which is incorporated by reference in its entirety.

Compositions for in vivo administration may be sterile. This can be easily done by filtration, for example, through sterile filtration membranes.

g. Use and method

The present disclosure relates to an improved method for generating universal antitoxic liquiritigenin. According to this improved method, phage display technology provides an alternative tool for selecting phage-expressed peptides that can bind many different venom targets with high specificity and affinity.

The novel methods disclosed herein produce antitoxicins with lower production costs, shorter synthesis times and fewer adverse reactions than any of the antitoxicin production methods known in the prior art. In addition, the antitoxic liquid produced by the present method is stable to long term storage in liquid form at ambient temperatures, a feature previously thought to be impossible for antitoxic liquids.

h. Detection & diagnostic uses

Their amino acid sequence, inhibition and cross-species reactivity were evaluated.

In addition to the universal antitoxicins, this novel method does not require helper phage, vector recloning and additional single chain fragment variable (scFv) antibody purification steps.

The peptides disclosed herein or compositions comprising the peptides disclosed herein can be used to determine the level of toxicity in a biological sample using classical immunohistological methods known to those skilled in the art, including immunoassays, such as enzyme-linked immunosorbent assays (ELISA), immunoprecipitations, or Western blots. Suitable antibody assay labels are known in the art and include enzyme labels, such as glucose oxidase; radioisotopes, e.g. iodine (A)¹²⁵I、¹²¹I) Carbon (C)¹⁴C) Sulfur (S), (S)³⁵S), tritium (³H) Indium (I) and (II)¹²¹In) and technetium (⁹⁹Tc); luminescent tags, such as luminol; and fluorescent labels such as fluorescein and rhodamine, and biotin. Such labels may be used to label the peptides disclosed herein or compositions comprising the peptides.

Determination of a detectable level of venom is intended to encompass qualitatively or quantitatively measuring or estimating the level of venom in a first biological sample, either directly (e.g., by determining or estimating an absolute level of venom) or relatively (e.g., by comparison to a disease-associated level of venom in a second biological sample). The level of venom in the first biological sample can be measured or estimated and compared to a standard level of venom taken from a second biological sample obtained from an individual not exposed to a noxious bite or determined by averaging levels from a population of individuals not exposed to venom. As understood in the art, once a "standard" venom level is known, it can be reused as a comparative standard.

The term "biological sample" as used herein refers to any biological sample obtained from a subject, cell line, tissue, or other cellular source potentially expressing a peptide disclosed herein. Methods for obtaining tissue biopsies and body fluids from animals (e.g., humans) are well known in the art. The biological sample contains peripheral mononuclear blood cells.

The peptides disclosed herein or compositions comprising the peptides disclosed herein may be used for prognostic, diagnostic, monitoring and screening applications, including in vitro and in vivo applications that are well known and considered standard by those skilled in the art and based on the present specification. Prognostic, diagnostic, monitoring and screening assays and kits for in vitro assessment and evaluation of immune system status and/or immune responses are useful for predicting, diagnosing and monitoring to evaluate patient samples, including those known or suspected of being exposed to toxic bites, or to make an assessment as to an expected or desired immune system response or antigenic response.

In one embodiment, the peptides disclosed herein or compositions comprising the peptides disclosed herein may be used in immunohistochemistry of biopsy samples.

In another embodiment, a peptide disclosed herein or a composition comprising a peptide disclosed herein can be used to detect PLA₂The level of (c). The peptides disclosed herein or compositions comprising the peptides disclosed herein may carry a detectable or functional label. When using fluorescent labeling, currently available microscopy and Fluorescence Activated Cell Sorting (FACS) analysis or a combination of the two method procedures known in the art can be used to identify and quantify specific binding members. The peptides disclosed herein or compositions comprising the peptides disclosed herein may carry a fluorescent label. Exemplary fluorescent labels include, for example, reactive and conjugated probes such as aminocoumarin, fluorescein, and texas red, Alexa Fluor dyes, Cy dyes, and DyLight dyes. The peptides disclosed herein or compositions comprising the peptides disclosed herein may carry a radioactive label, such as an isotope³H、¹⁴C、³²P、³⁵S、³⁶Cl、⁵¹Cr、⁵⁷Co、⁵⁸Co、⁵⁹Fe、⁶⁷Cu、⁹⁰Y、⁹⁹Tc、¹¹¹In、¹¹⁷Lu、¹²¹I、¹²⁴I、¹²⁵I、¹³¹I、¹⁹⁸Au、²¹¹At、²¹³Bi、²²⁵Ac and¹⁸⁶re. When using radiolabels, currently available counting methods known in the art can be used to confirmAnd quantifying the peptides disclosed herein or a composition comprising the peptides disclosed herein with PLA₂Specific binding of (3). Where the label is an enzyme, detection may be accomplished by any currently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gas analytical technique as is known in the art. This may be achieved by allowing the peptides disclosed herein or compositions comprising the disclosed peptides and PLA₂Or a control sample with a peptide disclosed herein or a composition comprising a peptide disclosed herein under conditions such that a complex is formed. Detecting and comparing a peptide disclosed herein or a composition comprising a peptide disclosed herein and PLA in a sample and a control₂Any complex formed in between. The peptides disclosed herein or compositions comprising the peptides disclosed herein can also be used to purify PLA via immunoaffinity purification₂。

Also included herein are assay systems that can be prepared in the form of test kits for quantitative analysis of, for example, PLA₂The degree of presence of (c). The system or test kit may include a labeled component, such as a labeled antibody, and one or more additional immunochemical reagents. See, e.g., (h) below for more information about the kit.

i. Therapeutic uses and methods

In some embodiments, the peptide binds snake venom and neutralizes venom toxicity. In some embodiments, the peptide binds to PLA₂And neutralize venom toxicity. The M13 phage can also be cleared from the body without adverse effects that can occur with antibody-based antisera therapy (e.g., seropathy). The methods of the present disclosure can be implemented to quickly and easily custom design an antitoxic liquid that is universal to toxic animals of any particular arm (subset) and is stable under conditions that are not tolerated by antibody-based therapies.

The peptides disclosed herein or compositions comprising the peptides disclosed herein can be delivered to a subject by a variety of routes. In some embodiments, the peptide or composition is delivered via parenteral, intranasal, intratracheal, oral, intradermal, topical, intramuscular, intraperitoneal, transdermal, intravenous, intratumoral, conjunctival and subcutaneous routes. Pulmonary administration may also be employed, for example, by using an inhaler or nebulizer, and the formulation of the aerosol as a spray.

The amount of a peptide disclosed herein or a composition comprising a peptide disclosed herein that will be effective in treating and/or preventing a disorder will depend on the nature of the disease and can be determined by standard clinical techniques.

The precise dose employed in the composition will also depend on the route of administration, and the severity of the infection or disease caused thereby, and should be decided according to the judgment of the practitioner and each subject's circumstances. For example, the effective dose can also vary depending on the mode of administration, the target, the physiological state of the patient (including age, weight, and health), whether the patient is a human or an animal, other drugs being administered, or whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, but non-human mammals, including transgenic mammals, may also be treated. Therapeutic doses are optimally titrated to optimize safety and efficacy.

In certain embodiments, in vitro assays are employed to help identify optimal dosage ranges. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

For passive immunization using the peptides disclosed herein, the dosage range is about 0.0001 to 100mg/kg, more typically 0.01 to 15mg/kg of patient body weight. For example, the dose may be 1mg/kg body weight, 10mg/kg body weight, or in the range of 1-10mg/kg, or in other words 70mg or 700mg or in the range of 70-700mg for a 70kg patient, respectively. In some embodiments, the dose administered to the patient is from about 1mg/kg to about 20mg/kg of patient body weight.

An exemplary treatment regimen entails administration once or in repeated doses. The interval between single doses may be hourly, daily, weekly, monthly, every 3 months, every 6 months, or yearly.

j. Reagent kit

In some embodiments, provided herein are kits comprising one or more of the peptides disclosed herein. In a specific embodiment, provided herein is a pharmaceutical pack or kit comprising one or more containers filled with one or more components of a protein, nucleic acid, or pharmaceutical composition described herein, such as one or more proteins provided herein. In some embodiments, the kit contains a pharmaceutical composition described herein and any prophylactic or therapeutic agent, such as those described herein. Optionally accompanying such a container may be a notice (notice) in a format prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biologicals, which notice reflects approval by the agency of manufacture, use or sale for human administration.

In some embodiments, provided herein is a diagnostic kit for identifying the type and severity of a bite of a toxic animal. In some embodiments, the kit comprises (a) a plurality of peptides, each peptide targeting a sequence unique to one of a plurality of animal venoms; (b) a plurality of labeling molecules, each labeling molecule conjugated to a corresponding one of the plurality of peptides; and (c) an assay configured to detect the labeled molecules, thereby revealing the peptides bound to their respective targets.

In some embodiments, the present disclosure also provides a diagnostic kit for identifying the type and severity of a bite wound in a toxic animal. In some embodiments, the kit comprises a plurality of peptides. In some embodiments, the kit comprises a plurality of marker molecules. In some embodiments, each peptide in the kit targets the sequence of the venom of a unique species. In some embodiments, one or more peptides in the kit target sequences unique to the venom of the same species.

In some embodiments, the sample tested in the kit is blood. In a particular embodiment, the sample tested in the kit is human blood. In some embodiments, the sample is isolated and contacted with one or more peptides in the kit. In some embodiments, the peptide in the kit is labeled.

In some embodiments, the kit further comprises an assay configured to detect the marker molecules, thereby revealing the peptides bound to their respective targets in the blood. Thus, in some embodiments, the kit can detect which peptides bind to the target, and the extent of binding, to identify what animal species venom is found in the blood and the severity of the bite.

Also provided herein are kits useful in the above methods. In one embodiment, the kit comprises a protein described herein, preferably a purified protein, in one or more containers. In one embodiment, a kit comprises a composition comprising a protein described herein in one or more containers. In a specific embodiment, the kits described herein contain a substantially isolated protein as a control. In another specific embodiment, the kits described herein further comprise a control antibody that is not reactive with a universal venom antigen. In another specific embodiment, the kits described herein contain one or more elements for detecting binding of a protein or composition comprising a protein to a universal venom antigen (e.g., the protein can be conjugated to a detectable substrate, e.g., a fluorescent, enzymatic, radioactive, or luminescent compound; or an antibody that recognizes a protein disclosed herein can be conjugated to a detectable substrate). In particular embodiments, the kits provided herein can comprise a recombinantly produced or chemically synthesized protein disclosed herein. The universal venom antigens provided in the kit may also be attached to a solid support. In a more specific embodiment, the detection device of the kit comprises a solid support to which the universal venom antigen is attached. Such kits may also contain an anti-human or anti-mouse/rat antibody that is not attached to a reporter marker. In this embodiment, binding of the protein to the venom can be detected by binding of the reporter-labeled antibody.

The following examples are provided by way of illustration and not by way of limitation.