Patent Application
20210269504
The present technology relates generally to methods and compositions pertaining to amphiphilic proteins that self-assemble to form stable micelles. Such amphiphilic proteins and corresponding micelles are useful for the delivery of hydrophobic compounds for a variety of applications.
The following description is provided to assist the understanding of the reader. None of the information provided or references cited is admitted to be prior art to the compositions and methods disclosed herein.
Previous studies relating to the recombinant expression of amphiphilic proteins has heavily focused on using naturally self-assembling proteins such as the sunflower protein oleosin, hydrogel formation by leucine zipper proteins, or elastin-like proteins (ELPs), which consist of repeats of the sequence VPGXG (SEQ ID NO: 64). While these constructs can form a variety of 3D structures in vivio and in vitro, they are limited in their ability for functionalization and rely heavily on the use of proteins that are known to naturally self-assemble. Accordingly, there is a need for a more effective and efficient approach to producing amphiphilic proteins.
In one aspect, the present disclosure provides an amphiphilic fusion protein having a formula S/I—X—H1—H2, wherein S— is a solubilizing moiety, I— is an insolubilizing moiety, —X— is a peptide sequence comprising a proteolytic or chemical cleavage site, —H1— is a hydrophilic peptide, and —H2 is a hydrophobic peptide.
In one aspect, the present disclosure provides an amphiphilic fusion protein having a formula S—X—H1—H2, wherein S— is a solubilizing moiety, —X— is a peptide sequence comprising a proteolytic cleavage site, —H1— is a hydrophilic peptide, and —H2 is a hydrophobic peptide.
In one aspect, the present disclosure provides an amphiphilic fusion protein having a formula I—X—H1—H2, wherein I— is an insolubilizing moiety, —X— is a peptide sequence comprising a chemical cleavage site, —H1— is a hydrophilic peptide, and —H2 is a hydrophobic peptide.
In some embodiments, the —H1— comprises an intrinsically disordered peptide (IDP) sequence. In some embodiments, the IDP sequence comprises one or more polypeptide sequences from a human neurofilament protein, a San1 protein, an Hsp-33 protein, an E1A protein, a PhD protein, a Sic1 protein, a WASP protein, a p27 protein, a CREB protein, a PUP protein, or a LEA protein. In some embodiments, the IDP comprises a human neurofilament polypeptide sequence. In some embodiments, the human neurofilament polypeptide sequence comprises the amino acid sequence as set forth in SEQ ID NO: 2. In some embodiments, the IDPs of the present technology comprise the human neurofilament polypeptide sequence as set forth in SEQ ID NO: 69, or fragments thereof. In some embodiments, the IDP comprises repeats of the sequence (SPAEAK)n (SEQ ID NO: 3) or repeats of the sequence (SPAEAR)n (SEQ ID NO: 4), where n is an integer from 2 to 50. In some embodiments, the IDP comprises repeats of the sequence (SPAX1AX2). (SEQ ID NO: 53), where X1 and X2 are each any charged amino acid and n is an integer from 2 to 50.
In some embodiments, the —H2 comprises a hydrophobic polypeptide sequence comprising a tyrosine-rich amino acid sequence 5-20 residues in length. In some embodiments, the —H2 comprises a hydrophobic polypeptide sequence selected from the group consisting of: YGAYAQYVYIYAYWYL (SEQ ID NO: 5), YGAYAQYVYIYAYWYLYAYI (SEQ ID NO: 6), YGAYAQYVYIYAYWYLYAYIAVAL (SEQ ID NO: 54), WEAKLAKALAKALAKHLAKALAKALKACEA (SEQ ID NO: 7), YWCCA(X)a (SEQ ID NO: 8) where a is a number of any hydrophobic residue (X), YWXXVbAb (SEQ ID NO: 9) where b is an integer of 3 or greater and X is any hydrophobic residue, and YWA(X)c (SEQ ID NO: 10) where c is a number of any hydrophobic residue (X).
In some embodiments, the S— comprises one or more of a maltose binding protein (MBP) polypeptide sequence, a small ubiquitin-like modifier (SUMO) polypeptide sequence, a glutathione S-transferase (GST) polypeptide sequence, a SlyD polypeptide sequence, a NusA polypeptide sequence, a thioredoxin polypeptide sequence, a ubiquitin polypeptide sequence, or a T7 gene 10 polypeptide sequence. In some embodiments, the S— further comprises a polyhistidine tag (His-tag). In some embodiments, the S— comprises a MBP polypeptide sequence. In some embodiments, the S— comprises an amino acid sequence set forth in SEQ ID NO: 12.
In some embodiments, the —X— comprises a proteolytic cleavage site selected from a thrombin cleavage site, a tobacco etch virus (TEV) cleavage site, a 3C cleavage site, an enterokinase cleavage site, or a Factor Xa cleavage site. In some embodiments, the proteolytic cleavage site is a thrombin cleavage site comprising the polypeptide sequence LVPR (SEQ ID NO: 13).
In some embodiments, the I— comprises a ketosteroid isomerase polypepide sequence. In some embodiments, the I— comprises an amino acid sequence set forth in SEQ ID NO: 55.
In some embodiments, the —X— comprises a chemical cleavage site selected from a CNBr cleavage site that cleaves at a methionine residue or a 2-nitro-5-thiocyanobenzoic acid cleavage site that cleaves at a cysteine residue.
In some embodiments, the fusion protein further comprises a cell targeting peptide (-T-) between the —X— and the —H1—, such that the amphiphilic fusion protein has the formula S/I-X-T-H1—H2. In some embodiments, the fusion protein further comprises a cell targeting peptide (-T-) between the —X— and the —H1—, such that the amphiphilic fusion protein has the formula S—X-T-H1—H2. In some embodiments, the fusion protein further comprises a cell targeting peptide (-T-) between the —X— and the —H1—, such that the amphiphilic fusion protein has the formula I-X-T-H1—H2. In some embodiments, the -T- is selected from the group consisting of a chitin binding domain (CBD), a cancer cell-targeting peptide, and an antimicrobial peptide. In some embodiments, the -T- is a cancer cell-targeting peptide selected from the group consisting of a peptide targeting human head and neck solid tumors and having the amino acid sequence TSPLNIHNGQKL (SEQ ID NO: 18), a peptide targeting tumor neovasculature and having the amino acid sequence CGKRK (SEQ ID NO: 19), a peptide targeting breast carcinoma and having the amino acid sequence CGNKRTRGC (SEQ ID NO: 20), a peptide targeting prostate vasculature and having the amino acid sequence SMSIARL (SEQ ID NO: 21), a peptide targeting hepatocellular carcinoma cells and having the amino acid sequence FQHPSFI (SEQ ID NO: 22), a peptide targeting integrin receptor and having the amino acid sequence RGD (SEQ ID NO: 23), a peptide targeting tumor neovasculature and having the amino acid sequence NGR (SEQ ID NO: 24), a peptide targeting endothelial VCAM-1 expressing cells and having the amino acid sequence VHSPNKK (SEQ ID NO: 25), a peptide targeting adenocarcinoma cells and having the amino acid sequence RRPYIL (SEQ ID NO: 26), a peptide targeting various carcinoma and having the amino acid sequence EDYELMDLLAYL (SEQ ID NO: 27), a peptide targeting breast carcinoma and having the amino acid sequence LTVSPWY (SEQ ID NO: 28), and a peptide targeting tumor neovasculature and having the amino acid sequence ATWLPPR (SEQ ID NO: 29). In some embodiments, the -T- is an antimicrobial peptide selected from the group consisting of a dermcidin, an apidaecin, a bactenecin, and a pyrrhocoricin. In some embodiments, the dermcidin is a dermcidin variant selected from the group consisting of DCD-1L comprising the amino acid sequence SSLLEKGLDGAKKAVGGLGKLGKDAVEDLESVGKGAVHDVKDVLDSVL (SEQ ID NO: 30), DCD-1 comprising the amino acid sequence SSLLEKGLDGAKKAVGGLGKLGKDAVEDLESVGKGAVHDVKDVLDSV (SEQ ID NO: 31), and SSL25 comprising the amino acid sequence SSLLEKGLDGAKKAVGGLGKLGKDA (SEQ ID NO: 32). In some embodiments, the apidaecin comprises the amino acid sequence GNNRP(V/I)YIPQPRPPHPR(L/I) (SEQ ID NO: 33). In some embodiments, the bactenecin is bactenecin 5 (Bac 5) or bactenecin 7 (Bac 7). In some embodiments, the pyrrhocoricin comprises the amino acid sequence
In some embodiments, the —H1—H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 56, and SEQ ID NO: 57.
In one aspect, the present disclosure provides an expression vector comprising a chimeric nucleic acid sequence encoding an amphiphilic fusion protein having a formula S/I—X—H1—H2, wherein S— is a solubilizing moiety, I— is an insolubilizing moiety, —X— is a peptide sequence comprising a proteolytic or chemical cleavage site, —H1— is a hydrophilic peptide, and —H2 is a hydrophobic peptide. In one aspect, the present disclosure provides an expression vector comprising a chimeric nucleic acid sequence encoding an amphiphilic fusion protein having a formula S—X—H1—H2, wherein S— is a solubilizing moiety, —X— is a peptide sequence comprising a proteolytic cleavage site, —H1— is a hydrophilic peptide, and —H2 is a hydrophobic peptide. In one aspect, the present disclosure provides an expression vector comprising a chimeric nucleic acid sequence encoding an amphiphilic fusion protein having a formula I—X—H1—H2, wherein I— is an insolubilizing moiety, —X— is a peptide sequence comprising a chemical cleavage site, —H1— is a hydrophilic peptide, and —H2 is a hydrophobic peptide.
In one aspect, the present disclosure provides a recombinant host cell engineered to express an amphiphilic fusion protein having a formula S/I—X—H1—H2, wherein S— is a solubilizing moiety, I— is an insolubilizing moiety, —X— is a peptide sequence comprising a proteolytic or chemical cleavage site, —H1— is a hydrophilic peptide, and —H2 is a hydrophobic peptide, wherein the host cell is a eukaryotic, prokaryotic, archaea, mammalian, yeast, bacteria, cyanobacteria, insect, or plant cell. In one aspect, the present disclosure provides a recombinant host cell engineered to express an amphiphilic fusion protein having a formula S—X—H1—H2, wherein S— is a solubilizing moiety, —X— is a peptide sequence comprising a proteolytic cleavage site, —H1— is a hydrophilic peptide, and —H2 is a hydrophobic peptide, wherein the host cell is a eukaryotic, prokaryotic, archaea, mammalian, yeast, bacteria, cyanobacteria, insect, or plant cell. In one aspect, the present disclosure provides a recombinant host cell engineered to express an amphiphilic fusion protein having a formula I-X—H1—H2, wherein I— is an insolubilizing moiety, —X— is a peptide sequence comprising a chemical cleavage site, —H1— is a hydrophilic peptide, and —H2 is a hydrophobic peptide, wherein the host cell is a eukaryotic, prokaryotic, archaea, mammalian, yeast, bacteria, cyanobacteria, insect, or plant cell. In some embodiments, the bacteria cell is E. coli.
In one aspect, the present disclosure provides a method of producing an amphiphilic fusion protein that spontaneously self-assembles to form a stable micelle, the method comprising: (a) introducing into a host cell an expression vector comprising a chimeric nucleic acid construct comprising, in the 5′ to 3′ direction, a promoter suitable for directing expression in a host cell operably linked to a nucleic acid sequence encoding an amphiphilic fusion protein having Formula (I): S/I—X—H1—H2, wherein S— is a solubilizing moiety, I— is an insolubilizing moiety, —X— is a peptide sequence comprising a proteolytic or chemical cleavage site, —H1— is a hydrophilic peptide, and —H2 is a hydrophobic peptide; (b) growing the host cell under conditions that allow for expression of the chimeric nucleic acid to produce the amphiphilic fusion protein; (c) purifying the amphiphilic fusion protein; and (d) contacting the amphiphilic fusion protein with a protease or a reagent to induce chemical cleavage to provide an amphiphilic fusion protein having Formula (II): H1—H2.
In some embodiments of the method, the chimeric nucleic acid construct of part (a) encodes an amphiphilic fusion protein further comprising a cell targeting peptide (-T-) between the —X— and the —H1—, such that the amphiphilic fusion protein has Formula (III): S/I-X-T-H1—H2, and such that after part (d) the amphiphilic fusion protein has Formula (IV): T-H1—H2.
In some embodiments of the method, the —H1— comprises an intrinsically disordered peptide (IDP) sequence. In some embodiments, the IDP sequence comprises one or more polypeptide sequences from a human neurofilament protein, a San1 protein, an Hsp-33 protein, an E1A protein, a PhD protein, a Sic1 protein, a WASP protein, a p27 protein, a CREB protein, a PUP protein, or a LEA protein. In some embodiments, the IDP comprises a human neurofilament polypeptide sequence. In some embodiments, the human neurofilament polypeptide sequence comprises the amino acid sequence as set forth in SEQ ID NO: 2. In some embodiments, the IDPs of the present technology comprise the human neurofilament polypeptide sequence as set forth in SEQ ID NO: 69, or fragments thereof. In some embodiments, the IDP comprises repeats of the sequence (SPAEAK)n (SEQ ID NO: 3) or repeats of the sequence (SPAEAR)n (SEQ ID NO: 4), where n is an integer from 2 to 50. In some embodiments, the IDP comprises repeats of the sequence (SPAX1AX2)n (SEQ ID NO:53), where X1 and X2 are each any charged amino acid and n is an integer from 2 to 50.
In some embodiments of the method, the —H2 comprises a hydrophobic polypeptide sequence comprising a tyrosine-rich amino acid sequence 5-20 residues in length. In some embodiments, the —H2 comprises a hydrophobic polypeptide sequence selected from the group consisting of: YGAYAQYVYIYAYWYL (SEQ ID NO: 5), YGAYAQYVYIYAYWYLYAYI (SEQ ID NO: 6), YGAYAQYVYIYAYWYLYAYIAVAL (SEQ ID NO: 54), WEAKLAKALAKALAKHLAKALAKALKACEA (SEQ ID NO: 7), YWCCA(X)a (SEQ ID NO: 8) where a is a number of any hydrophobic residue (X), YWXXVbAb (SEQ ID NO: 9) where b is an integer of 3 or greater and X is any hydrophobic residue, and YWA(X)c (SEQ ID NO: 10) where c is a number of any hydrophobic residue (X).
In some embodiments of the method, the S— comprises one or more of a maltose binding protein (MBP) polypeptide sequence, a small ubiquitin-like modifier (SUMO) polypeptide sequence, a glutathione S-transferase (GST) polypeptide sequence, a SlyD polypeptide sequence, a NusA polypeptide sequence, a thioredoxin polypeptide sequence, a ubiquitin polypeptide sequence, or a T7 gene 10 polypeptide sequence. In some embodiments, the S— further comprises a polyhistidine tag (His-tag). In some embodiments, the S— comprises a MBP polypeptide sequence. In some embodiments, the S— comprises an amino acid sequence set forth in SEQ ID NO: 12.
In some embodiments of the method, the —X— comprises a proteolytic cleavage site selected from a thrombin cleavage site, a tobacco etch virus (TEV) cleavage site, a 3C cleavage site, an enterokinase cleavage site, or a Factor Xa cleavage site. In some embodiments, the proteolytic cleavage site is a thrombin cleavage site comprising the polypeptide sequence LVPR (SEQ ID NO: 13).
In some embodiments, the I— comprises a ketosteroid isomerase polypepide sequence. In some embodiments, the I— comprises an amino acid sequence set forth in SEQ ID NO: 55.
In some embodiments, the —X— comprises a chemical cleavage site selected from a CNBr cleavage site that cleaves at a methionine residue or a 2-nitro-5-thiocyanobenzoic acid cleavage site that cleaves at a cysteine residue.
In some embodiments of the method, the -T- is selected from the group consisting of a chitin binding domain (CBD), a cancer cell-targeting peptide, and an antimicrobial peptide. In some embodiments, the -T- is a cancer cell-targeting peptide selected from the group consisting of a peptide targeting human head and neck solid tumors and having the amino acid sequence TSPLNIHNGQKL (SEQ ID NO: 18), a peptide targeting tumor neovasculature and having the amino acid sequence CGKRK (SEQ ID NO: 19), a peptide targeting breast carcinoma and having the amino acid sequence CGNKRTRGC (SEQ ID NO: 20), a peptide targeting prostate vasculature and having the amino acid sequence SMSIARL (SEQ ID NO: 21), a peptide targeting hepatocellular carcinoma cells and having the amino acid sequence FQHPSFI (SEQ ID NO: 22), a peptide targeting integrin receptor and having the amino acid sequence RGD (SEQ ID NO: 23), a peptide targeting tumor neovasculature and having the amino acid sequence NGR (SEQ ID NO: 24), a peptide targeting endothelial VCAM-1 expressing cells and having the amino acid sequence VHSPNKK (SEQ ID NO: 25), a peptide targeting adenocarcinoma cells and having the amino acid sequence RRPYIL (SEQ ID NO: 26), a peptide targeting various carcinoma and having the amino acid sequence EDYELMDLLAYL (SEQ ID NO: 27), a peptide targeting breast carcinoma and having the amino acid sequence LTVSPWY (SEQ ID NO: 28), and a peptide targeting tumor neovasculature and having the amino acid sequence ATWLPPR (SEQ ID NO: 29). In some embodiments, the -T- is an antimicrobial peptide selected from the group consisting of a dermcidin, an apidaecin, a bactenecin, and a pyrrhocoricin. In some embodiments, the dermcidin is a dermcidin variant selected from the group consisting of DCD-1L comprising the amino acid sequence SSLLEKGLDGAKKAVGGLGKLGKDAVEDLESVGKGAVHDVKDVLDSVL (SEQ ID NO: 30), DCD-1 comprising the amino acid sequence SSLLEKGLDGAKKAVGGLGKLGKDAVEDLESVGKGAVHDVKDVLDSV (SEQ ID NO: 31), and SSL25 comprising the amino acid sequence SSLLEKGLDGAKKAVGGLGKLGKDA (SEQ ID NO: 32). In some embodiments, the apidaecin comprises the amino acid sequence GNNRP(V/I)YIPQPRPPHPR(L/I) (SEQ ID NO: 33). In some embodiments, the bactenecin is bactenecin 5 (Bac 5) or bactenecin 7 (Bac 7). In some embodiments, the pyrrhocoricin comprises the amino acid sequence
In some embodiments of the method, the —H1—H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 56, and SEQ ID NO: 57.
In one aspect, the present disclosure provides a micelle comprising an amphiphilic fusion protein comprising: (i) a hydrophilic peptide (H1); and (ii) a hydrophobic peptide (H2).
In some embodiments, the H1 comprises an intrinsically disordered peptide (IDP) sequence. In some embodiments, the IDP sequence comprises one or more polypeptide sequences from a human neurofilament protein, a San1 protein, an Hsp-33 protein, an E1A protein, a PhD protein, a Sic1 protein, a WASP protein, a p27 protein, a CREB protein, a PUP protein, or a LEA protein. In some embodiments, the IDP comprises a human neurofilament polypeptide sequence. In some embodiments, the human neurofilament polypeptide sequence comprises the amino acid sequence as set forth in SEQ ID NO: 2. In some embodiments, the IDPs of the present technology comprise the human neurofilament polypeptide sequence as set forth in SEQ ID NO: 69, or fragments thereof. In some embodiments, the IDP comprises repeats of the sequence (SPAEAK)n (SEQ ID NO: 3) or repeats of the sequence (SPAEAR)n (SEQ ID NO: 4), where n is an integer from 2 to 50. In some embodiments, the IDP comprises repeats of the sequence (SPAX1AX2)n (SEQ ID NO: 53), where X1 and X2 are each any charged amino acid and n is an integer from 2 to 50.
In some embodiments, the H2 comprises a hydrophobic polypeptide sequence comprising a tyrosine-rich amino acid sequence 5-20 residues in length. In some embodiments, the H2 comprises a hydrophobic polypeptide sequence selected from the group consisting of: YGAYAQYVYIYAYWYL (SEQ ID NO: 5), YGAYAQYVYIYAYWYLYAYI (SEQ ID NO: 6), WEAKLAKALAKALAKHLAKALAKALKACEA (SEQ ID NO: 7), YWCCA(X)a (SEQ ID NO: 8) where a is a number of any hydrophobic residue (X), YWXXVbAb (SEQ ID NO: 9) where b is an integer of 3 or greater and X is any hydrophobic residue, and YWA(X)c (SEQ ID NO: 10) where c is a number of any hydrophobic residue (X).
In some embodiments, the amphiphilic fusion protein further comprises a cell targeting peptide (T) covalently linked to the N-terminus of the H1. In some embodiments, the T is selected from the group consisting of a chitin binding domain (CBD), a cancer cell-targeting peptide, and an antimicrobial peptide. In some embodiments, the cancer cell-targeting peptide is selected from the group consisting of a peptide targeting human head and neck solid tumors and having the amino acid sequence TSPLNIHNGQKL (SEQ ID NO: 18), a peptide targeting tumor neovasculature and having the amino acid sequence CGKRK (SEQ ID NO: 19), a peptide targeting breast carcinoma and having the amino acid sequence CGNKRTRGC (SEQ ID NO: 20), a peptide targeting prostate vasculature and having the amino acid sequence SMSIARL (SEQ ID NO: 21), a peptide targeting hepatocellular carcinoma cells and having the amino acid sequence FQHPSFI (SEQ ID NO: 22), a peptide targeting integrin receptor and having the amino acid sequence RGD (SEQ ID NO: 23), a peptide targeting tumor neovasculature and having the amino acid sequence NGR (SEQ ID NO: 24), a peptide targeting endothelial VCAM-1 expressing cells and having the amino acid sequence VHSPNKK (SEQ ID NO: 25), a peptide targeting adenocarcinoma cells and having the amino acid sequence RRPYIL (SEQ ID NO: 26), a peptide targeting various carcinoma and having the amino acid sequence EDYELMDLLAYL (SEQ ID NO: 27), a peptide targeting breast carcinoma and having the amino acid sequence LTVSPWY (SEQ ID NO: 28), and a peptide targeting tumor neovasculature and having the amino acid sequence ATWLPPR (SEQ ID NO: 29). In some embodiments, the T is an antimicrobial peptide selected from the group consisting of a dermcidin, an apidaecin, a bactenecin, and a pyrrhocoricin. In some embodiments, the dermcidin is a dermcidin variant selected from the group consisting of DCD-1L comprising the amino acid sequence SSLLEKGLDGAKKAVGGLGKLGKDAVEDLESVGKGAVHDVKDVLDSVL (SEQ ID NO: 30), DCD-1 comprising the amino acid sequence SSLLEKGLDGAKKAVGGLGKLGKDAVEDLESVGKGAVHDVKDVLDSV (SEQ ID NO: 31), and SSL25 comprising the amino acid sequence SSLLEKGLDGAKKAVGGLGKLGKDA (SEQ ID NO: 32). In some embodiments, the apidaecin comprises the amino acid sequence GNNRP(V/I)YIPQPRPPHPR(L/I) (SEQ ID NO: 33). In some embodiments, the bactenecin is bactenecin 5 (Bac 5) or bactenecin 7 (Bac 7). In some embodiments, the pyrrhocoricin comprises the amino acid sequence
In some embodiments, the critical micelle concentration (CMC) of the amphiphilic fusion protein in water is from about 10 μM to about 20 μM at a physiological pH of about 7.4.
In some embodiments, the micelle has a diameter from about 20 nm to about 40 nm. In some embodiments, the micelle has a diameter of about 27 nm.
In some embodiments, the micelle is stable at a pH from about 2.0 to about 10.0.
In some embodiments, the micelle is stable at a temperature from about 25° C. to about 70° C.
In some embodiments, the micelle further comprises a fluorescent dye. In some embodiments, the fluorescent dye is covalently attached to the hydrophilic peptide (H1). In some embodiments, the fluorescent dye is covalently attached to the hydrophobic peptide (H2). In some embodiments, the fluorescent dye is fluorescein or rhodamine.
In some embodiments, the micelle has a core-shell structure. In some embodiments, the micelle has a shell diameter from about 40 nm to about 75 nm. In some embodiments, the micelle has a core diameter from about 25 nm to about 45 nm. In some embodiments, the micelle has a shell thickness from about 5 nm to about 20 nm.
In some embodiments, the micelle further comprises a hydrophobic cargo. In some embodiments, the hydrophobic cargo is a drug, a fungicide, a protein, a nucleic acid, a hormone, a receptor, a diagnostic agent, an imaging agent, a metal complex, a silicone oil, a triglyceride, or a combination thereof.
In some embodiments, the amphiphilic fusion protein comprising H1 and H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 56, and SEQ ID NO: 57.
In one aspect, the preset disclosure provides a pharmaceutical composition comprising the micelle of the present technology and a hydrophobic cargo, wherein the hydrophobic cargo is a therapeutically active agent.
In one aspect, the preset disclosure provides a method for treating a disease or disorder in a subject in need thereof comprising administering the pharmaceutical composition to the subject.
In one aspect, the preset disclosure provides a composition suitable for use in drug delivery, cosmetics, paints and coatings, crop protection, nanoparticle synthesis and catalysis, home and personal care, and cleaning, comprising the micelle of the present technology.
It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present technology are described below in various levels of detail in order to provide a substantial understanding of the present technology. The definitions of certain terms as used in this specification are provided below. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this present technology belongs.
The following terms are used herein, the definitions of which are provided for guidance.
As used herein, the singular forms “a,” “an,” and “the” designate both the singular and the plural, unless expressly stated to designate the singular only.
The term “about” and the use of ranges in general, whether or not qualified by the term about, means that the number comprehended is not limited to the exact number set forth herein, and is intended to refer to ranges substantially within the quoted range while not departing from the scope of the present technology. As used herein, “about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” will mean up to plus or minus 10% of the particular term.
The term “administered” or “administration” when used in the context of therapeutic and diagnostic uses, refers to and includes the introduction of a selected amount of the micelles described herein into an in vivo or in vitro environment for the purpose of, for example, delivering a therapeutic agent to a targeted site. Administration can be carried out by any suitable route, including but not limited to, intravenously, intramuscularly, intraperitoneally, subcutaneously, and other suitable routes as described herein. Administration includes self-administration and the administration by another.
As used herein, “amphiphilic fusion protein” refers to a protein created by the joining of translational sequences from two or more different genes to create one contiguous hybrid or chimeric protein molecule comprising a hydrophobic domain in translational fusion with a hydrophilic domain. The amphiphilic fusion proteins of the present technology may also comprise a solubilizing domain and a proteolytic cleavage site in translational fusion with the hydrophobic domain. In some embodiments, the amphiphilic fusion proteins of the present technology further comprise a cell targeting peptide in translational fusion with the hydrophobic domain. In some embodiments, “amphiphilic fusion protein” refers to micelles comprising the amphiphilic fusion proteins.
The term “cell targeting peptide” refers to a peptide that is conventionally used in the art to recognize and bind specific cells and tissues. In some embodiments, the amphiphilic fusion peptides of the present technology, which form stable micelles, may be conjugated to one or more cell targeting peptides to achieve targeted delivery of an agent or hydrophobic cargo to specific cells and tissues.
A “chimeric nucleic acid” comprises a coding sequence or fragment thereof linked to a nucleotide sequence that is different from the nucleotide sequence with which it is associated in cells in which the coding sequence occurs naturally.
As used herein, the terms “effective amount” or “therapeutically effective amount” or “pharmaceutically effective amount” refer to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount which results in the prevention of, disease, condition and/or symptom(s) thereof. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to the composition drugs. It will also depend on the degree, severity and type of disease or condition. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. In some embodiments, multiple doses are administered. Additionally or alternatively, in some embodiments, multiple therapeutic compositions or compounds are administered.
“Heterologous nucleic acid” refers to a nucleic acid, DNA, or RNA, which has been introduced into a cell, and which is not a copy of a sequence naturally found in the cell into which it is introduced. Such heterologous nucleic acid may comprise segments that are a copy of a sequence that is naturally found in the cell into which it has been introduced, or fragments thereof.
As used herein, a recombinant or engineered “host cell” refers to a cell e.g., eukaryotic, prokaryotic, yeast, bacteria, such as Escherichia coli, cyanobacteria, insect, plant, archaea, cell-free, or mammalian cell, that has been modified such that it produces fusion proteins of the present technology. In some embodiments, the host cells are in vitro, cultured cells. In some embodiments, the recombinant host cell comprises one or more polynucleotides, each polynucleotide encoding an amphiphilic fusion protein of the present technology or portions thereof.
As used herein, “hydrophobic cargo” refers to any hydrophobic compound or agent that is suitable for delivery by the micelles described herein. Examples of suitable hydrophobic cargo include but are not limited to a drug, a fungicide, a protein, a nucleic acid, a hormone, a receptor, a diagnostic agent, an imaging agent, a metal complex, a silicone oil, a triglyceride, or a combination thereof. Hydrophobic cargo may include hydrophobic agents that are biologically and/or pharmaceutically active.
As used herein, “insolubilizing moiety” refers to a moiety, such as a peptide, that enhances the insolubility of the amphiphilic proteins described herein and in some instances, prevents the amphiphilic protein from undergoing self-assembly to form a micelle. In some embodiments, the insolubilizing moiety comprises a ketosteroid isomerase polypeptide sequence. In some embodiments, the insolubilizing moiety comprises an amino acid sequence as set for in SEQ ID NO: 55. In some embodiments, the insolubilizing moiety is a peptide that further contains a chemical cleavage site and is cleavable. In some embodiments, the chemical cleavage site selected from a CNBr (cyanogen bromide) cleavage site that cleaves at a methionine residue or a 2-nitro-5-thiocyanobenzoic acid cleavage site that cleaves at a cysteine residue.
As used herein, “intrinsically disordered proteins (IDPs), also known as intrinsically unstructured proteins (IUPs), are characterized by the lack of a stable tertiary structure under physiological conditions. In some embodiments, the IDP comprises a polypeptide sequence selected from a human neurofilament protein, San1 protein, Hsp-33 protein, E1A protein, PhD protein, Sic1 protein, WASP protein, p27 protein, CREB protein, PUP protein, LEA protein, or portions or fragments thereof containing intrinsically disordered regions. In some embodiments, the IDPs of the present technology comprise the human neurofilament polypeptide sequence as set forth in SEQ ID NO: 2, or fragments thereof. In some embodiments, the IDPs of the present technology comprise the human neurofilament polypeptide sequence as set forth in SEQ ID NO: 69, or fragments thereof. In some embodiments, the IDPs of the present technology comprise repeats of the sequence (SPAEAK)n (SEQ ID NO: 3), where n is an integer from 2 to 100, or any range in between, such as 2 to 50, or 2 to 25. In some embodiments, n is 25. In some embodiments, the IDPs of the present technology comprise repeats of the sequence (SPAEAR)d (SEQ ID NO: 4), where d is an integer from 2 to 100, or any range in between, such as 2 to 50, or 2 to 25. In some embodiments, d is 25. In some embodiments, the IDP comprises repeats of the sequence (SPAX1AX2)n (SEQ ID NO: 53), where X1 and X2 are each any charged amino acid and n is an integer from 2 to 100, or any range in between, such as 2 to 50, or 2 to 25. In some embodiments, n is 25.
As used herein, the term “purify,” “purified,” or “purification” means the removal or isolation of a molecule from its environment by, for example, isolation or separation.
As used herein, the term “recombinant polypeptide” refers to a polypeptide that is produced by recombinant DNA techniques, wherein generally DNA encoding the expressed protein or RNA is inserted into a suitable expression vector and that is in turn used to transform a host cell to produce the polypeptide or RNA.
As used herein, “solubilizing moiety” refers to a moiety, such as a peptide, that enhances the solubility of the amphiphilic proteins described herein and in some instances, prevents the amphiphilic protein from undergoing self-assembly to form a micelle. In some embodiments, the solubilizing moiety comprises one or more of a maltose binding protein (MBP) polypeptide sequence, a small ubiquitin-like modifier (SUMO) polypeptide sequence, a glutathione S-transferase (GST) polypeptide sequence, a SlyD polypeptide sequence, a NusA polypeptide sequence, a thioredoxin polypeptide sequence, a ubiquitin polypeptide sequence, or a T7 gene 10 polypeptide sequence. In some embodiments, the solubilizing moiety further comprises a polyhistidine tag (His-tag), such as a 6×His tag. In some embodiments, the solubilizing moiety comprises an amino acid sequence as set for thein SEQ ID NO: 12. In some embodiments, the solubilizing moiety is a peptide that further contains a proteolytic cleavage site and is cleavable. In some embodiments, the proteolytic cleavage site is selected from a thrombin cleavage site (e.g., LVPR; SEQ ID NO: 13), a tobacco etch virus cleavage site (e.g., ENLYFQ; SEQ ID NO: 14), a 3C cleavage site (e.g., LEVLFQ; SEQ ID NO: 15), an enterokinase cleavage site (e.g., DDDDK; SEQ ID NO: 16), or a Factor Xa cleavage site (e.g., IEGR; SEQ ID NO: 17).
As used herein, the term “subject” refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like (e.g., which is to be the recipient of a particular treatment, or from whom cells are harvested).
The term “therapeutic active agent,” and similar terms referring to a therapeutic or medicinal function mean that the referenced small molecule, macromolecule, protein, nucleic acid, growth factor, hormone, drug, other substance, cell, metal complex, a silicone oil, a triglyceride, or combination thereof can beneficially affect the initiation, course, and/or one or more symptoms of a disease or condition in a subject, and may be used in conjunction with the micelles described herein in the manufacture of medicaments for treating a disease or other condition. Suitable therapeutic agents for encapsulation in the micelles described herein include hydrophobic therapeutic agents.
The terms “treating”, “treat” and “treatment” can include (i) preventing a disease, pathologic or medical condition from occurring (e.g., prophylaxis); (ii) inhibiting the disease, pathologic or medical condition or arresting its development; (iii) relieving the disease, pathologic or medical condition; and/or (iv) diminishing symptoms associated with the disease, pathologic or medical condition. Thus, the terms “treat”, “treatment”, and “treating” can extend to prophylaxis and can include prevent, prevention, preventing, lowering, stopping or reversing the progression or severity of the condition or symptoms being treated. As such, the term “treatment” can include medical, therapeutic, and/or prophylactic administration, as appropriate.
As used herein, the term “vector” or “expression vector” refers to a nucleic acid molecule capable of directing the expression of genes to which they are operatively linked. In general, expression vectors of utility in recombinant DNA techniques are often in the form of “plasmids,” which refer generally to circular double stranded DNA loops that, in their vector form, are not bound to the chromosome. The terms “plasmid” and “vector” are used interchangeably herein. The expression vectors described herein include a polynucleotide sequence described herein in a form suitable for expression of the polynucleotide sequence in a host cell. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc. The expression vectors described herein can be introduced into host cells to produce polypeptides, including fusion polypeptides such as amphiphilic fusion proteins, encoded by the polynucleotide sequences as described herein.
As used herein, “IDP1-2Yx2A” refers to SEQ ID NO: 39 or micelles comprising SEQ ID NO: 39, depending on the context in which it is used. In some embodiments, “IDP2-2Yx2A” refers to SEQ ID NO: 56 or micelles comprising SEQ ID NO: 56, depending on the context in which it is used. As used herein, “IDP-2Yx3A” refers to SEQ ID NO: 42 or micelles comprising SEQ ID NO: 42, depending on the context in which it is used. As used herein, “IDP-2Yx4A” refers to SEQ ID NO: 57 or micelles comprising SEQ ID NO: 57.
Recent efforts relating to the production of amphiphilic proteins that self-assemble have been focused on using naturally self-assembling proteins, which requires the use of proteins that are known to naturally self-assemble and limits the potential for further functionalization. Other approaches have focused on the use of polymers, small molecules, and peptides. These approaches are relatively cost-inefficient and laborious.
To address these shortcomings, the present technology relates to a series of biodegradable amphiphilic fusion proteins comprising an intrinsically disordered protein (IDP) segment that are produced through a biological mechanism. Accordingly, provided herein in one aspect are recombinant amphiphilic proteins that self-assemble to form stable micelles. The amphiphilic proteins of the instant disclosure contain a hydrophilic repetitive sequence derived from a naturally disordered protein (e.g., an intrinsically disordered protein (IDP)) and a designed hydrophobic region to allow for self-aggregation. Creating a self-assembling amphiphilic protein from a naturally disordered sequence provides an opportunity for further functionalization that has yet to be realized with methods for preparing amphiphilic proteins that require the use of naturally self-assembling proteins. Furthermore, this disclosure recognizes that genetically encoding a solubility enhancing and cleavable protein group to the amphiphilic proteins described herein provides an efficient method for producing self-assembling proteins in a controlled manner after expression and initial purification that is not achieved by expressing the amphiphilic protein alone.
In some embodiments, the micelles formed from the recombinant proteins described herein have desirable properties that render these micelles suitable for the delivery of a variety of hydrophobic agents in a myriad of applications. Such desirable properties include, but are not limited to, low critical micelle concentration (CMC), pH stability, temperature stability, encapsulation efficiency, size, potential for exterior modification, and biodegradability. Such applications, include but are not limited to, drug delivery, cosmetics, paints and coatings, crop protection, nanoparticle synthesis and catalysis, home and personal care, and cleaning.
The present technology provides compositions comprising an amphiphilic fusion protein comprising a hydrophilic peptide (H1) fused to a hydrophobic peptide (H2). In some embodiments the amphiphilic fusion protein comprises a solubilizing moiety (S) and a proteolytic cleavage site (X) fused to the N-terminus of the hydrophilic peptide. In some embodiments, the amphiphilic fusion proteins of the present technology have the general structure shown below:
S—X—H1—H2
In some embodiments, the amphiphilic fusion proteins further comprise a cell targeting peptide (T) between the proteolytic cleavage site (X) and the hydrophilic peptide (H1) and has the general structure shown below:
S—X-T-H1—H2
In some embodiments, the amphiphilic fusion proteins, after cleavage of the S—X domains, spontaneously self-assemble to form stable micelles.
In some embodiments the amphiphilic fusion protein comprises a insolubilizing moiety (I) and a chemical cleavage site (X) fused to the N-terminus of the hydrophilic peptide. In some embodiments, the amphiphilic fusion proteins of the present technology have the general structure shown below:
I—X—H1—H2.
In some embodiments, the amphiphilic fusion proteins further comprise a cell targeting peptide (T) between the chemical cleavage site (X) and the hydrophilic peptide (H1) and has the general structure shown below:
I-X-T-H1—H2
In some embodiments, the amphiphilic fusion proteins, after cleavage of the I-X domains, spontaneously self-assemble to form stable micelles.
A. Hydrophilic peptides (H1)
In some embodiments, the hydrophilic peptides of the present technology comprise an intrinsically disordered protein (IDP). In some embodiments, the IDP comprises a polypeptide sequence selected from a human neurofilament protein, San1 protein, Hsp-33 protein, E1A protein, PhD protein, Sic1 protein, WASP protein, p27 protein, CREB protein, PUP protein, LEA protein, or portions or fragments thereof containing intrinsically disordered regions. In some embodiments, the IDP comprises the entire protein or fragments of proteins containing intrinsically disordered peptide regions. In some embodiments, the IDPs of the present technology comprise the human neurofilament polypeptide sequence as set forth in SEQ ID NO: 2, or fragments thereof. In some embodiments, the IDPs of the present technology comprise the human neurofilament polypeptide sequence as set forth in SEQ ID NO: 69, or fragments thereof. Exemplary human neurofilament nucleic acid and polypeptide sequences are provided in Table 1.
In some embodiments, the IDPs of the present technology comprise repeats of the sequence (SPAEAK)n (SEQ ID NO: 3), where n is an integer from 2 to 100, or any range in between, such as 2 to 50, or 2 to 25. In some embodiments, n is 25. In some embodiments, the IDPs of the present technology comprise repeats of the sequence (SPAEAR)d (SEQ ID NO: 4), where d is an integer from 2 to 100, or any range in between, such as 2 to 50, or 2 to 25. In some embodiments, d is 25. In some embodiments, the IDP comprises repeats of the sequence (SPAX1AX2)n (SEQ ID NO: 53), where X1 and X2 are each any charged amino acid and n is an integer from 2 to 100, or any range in between, such as 2 to 50, or 2 to 25. In some embodiments, n is 25.
B. Hydrophobic Peptides (H2)
In some embodiments, the hydrophobic peptides of the present technology comprise a hydrophobic polypeptide sequence selected from the group consisting of: YGAYAQYVYIYAYWYL (SEQ ID NO: 5), YGAYAQYVYIYAYWYLYAYI (SEQ ID NO: 6), WEAKLAKALAKALAKHLAKALAKALKACEA (SEQ ID NO: 7), YWCCA(X)a (SEQ ID NO: 8) where a is a number of any hydrophobic residue (X), YWXXVbAb (SEQ ID NO: 9) where b is an integer of 3 or greater and X is any hydrophobic residue, and YWA(X)c (SEQ ID NO: 10) where c is a number of any hydrophobic residue (X).
C. Solubilizing Moieties (S)/Insolubilizing Moieties (I)
In some embodiments, the solubilizing moieties of the present technology comprise one or more of a maltose binding protein (MBP) polypeptide sequence, a small ubiquitin-like modifier (SUMO) polypeptide sequence, a glutathione S-transferase (GST) polypeptide sequence, a SlyD polypeptide sequence, a NusA polypeptide sequence, a thioredoxin polypeptide sequence, a ubiquitin polypeptide sequence, or a T7 gene 10 polypeptide sequence. In some embodiments, the solubilizing moiety further comprises a polyhistidine tag (His-tag), such as a 6×His tag. An exemplary nucleic acid sequence for an MBP and its polypeptide sequence are set forth in Table 2A.
In some embodiments, the insolubilizing moieties of the present technology comprise a ketosteroid isomerase (KSI) polypeptide sequence. An exemplary nucleic acid sequence for a KSI and its polypeptide sequence are set forth in Table 2B.
D. Proteolytic Cleavage Sites/Chemical Cleavage Sites (X)
Exemplary, non-limiting proteolytic cleavage sites comprise a thrombin cleavage site (e.g., LVPR; SEQ ID NO: 13), a tobacco etch virus cleavage site (e.g., ENLYFQ; SEQ ID NO: 14), a 3C cleavage site (e.g., LEVLFQ; SEQ ID NO: 15), an enterokinase cleavage site (e.g., DDDDK; SEQ ID NO: 16), or a Factor Xa cleavage site (e.g., IEGR; SEQ ID NO: 17).
Exemplary, non-limiting chemical cleavage sites comprise a chemical cleavage site selected from a CNBr (cyanogen bromide) cleavage site that cleaves at a methionine residue or a 2-nitro-5-thiocyanobenzoic acid cleavage site that cleaves at a cysteine residue.
E. Cell Targeting Peptides (T)
In some embodiments, the amphiphilic fusion proteins further comprise a cell targeting peptide (T) that can be used to specifically target the amphiphilic fusion proteins or micelles comprising the amphiphilic fusion proteins to a particular cell or tissue. In some embodiments, the cell targeting peptides are useful in methods for delivering hydrophobic cargo to the interior of target cells (e.g., cancer cells, fungal cells, microbial cells).
Thus, in some embodiments, the cell targeting peptide is a cancer cell-targeting peptide selected from the group consisting of a peptide targeting human head and neck solid tumors and having the amino acid sequence TSPLNIHNGQKL (SEQ ID NO: 18), a peptide targeting tumor neovasculature and having the amino acid sequence CGKRK (SEQ ID NO: 19), a peptide targeting breast carcinoma and having the amino acid sequence CGNKRTRGC (SEQ ID NO: 20), a peptide targeting prostate vasculature and having the amino acid sequence SMSIARL (SEQ ID NO: 21), a peptide targeting hepatocellular carcinoma cells and having the amino acid sequence FQHPSFI (SEQ ID NO: 22), a peptide targeting integrin receptor and having the amino acid sequence RGD (SEQ ID NO: 23), a peptide targeting tumor neovasculature and having the amino acid sequence NGR (SEQ ID NO: 24), a peptide targeting endothelial VCAM-1 expressing cells and having the amino acid sequence VHSPNKK (SEQ ID NO: 25), a peptide targeting adenocarcinoma cells and having the amino acid sequence RRPYIL (SEQ ID NO: 26), a peptide targeting various carcinoma and having the amino acid sequence EDYELMDLLAYL (SEQ ID NO: 27), a peptide targeting breast carcinoma and having the amino acid sequence LTVSPWY (SEQ ID NO: 28), and a peptide targeting tumor neovasculature and having the amino acid sequence ATWLPPR (SEQ ID NO: 29).
In some embodiments, the cell targeting peptide comprises a chitin binding domain (CBD) that targets fungal cells.
In some embodiments, the cell targeting peptide comprises an antimicrobial peptide that targets microbes. In some embodiments, the antimicrobial peptide is selected from the group consisting of a dermcidin, an apidaecin, a bactenecin, and a pyrrhocoricin. In some embodiments, the dermcidin is a dermcidin variant selected from the group consisting of DCD-1L comprising the amino acid sequence SSLLEKGLDGAKKAVGGLGKLGKDAVEDLESVGKGAVHDVKDVLDSVL (SEQ ID NO: 30), DCD-1 comprising the amino acid sequence SSLLEKGLDGAKKAVGGLGKLGKDAVEDLESVGKGAVHDVKDVLDSV (SEQ ID NO: 31), and SSL25 comprising the amino acid sequence SSLLEKGLDGAKKAVGGLGKLGKDA (SEQ ID NO: 32). In some embodiments, the apidaecin comprises the amino acid sequence GNNRP(V/I)YIPQPRPPHPR(L/I) (SEQ ID NO: 33). In some embodiments, the bactenecin is bactenecin 5 (Bac 5) or bactenecin 7 (Bac 7). In some embodiments, the pyrrhocoricin comprises the amino acid sequence
F. Recombinant Fusion Protein Nucleic Acid and Amino Acid Sequences
Exemplary IDP-Maltose binding protein (MBP) nucleic acid and amino acid sequences are provided in Table 3A.
AAGAGTCCAGCGGAAGCTAAGTCGCCAGCCGAAGTCAAGTCGCCCGC
CGTCGCGAAAAGCCCCGCAGAGGTGAAATCCCCGGCCGAAGTCAAAT
CGCCGGCAGAAGCGAAATCCCCGGCAGAAGCAAAAAGTCCTGCTGAG
GTCAAATCGCCAGCAACCGTCAAATCCCCTGGAGAGGCAAAATCTCC
GGCAGAAGCCAAGTCCCCTGCCGAAGTGAAGTCACCTGTCGAAGCCA
AGTCGCCGGCCGAAGCGAAGAGCCCAGCGAGCGTGAAAAGTCCTGGT
GAGGCTAAGTCCCCGGCGGAAGCGAAATCTCCAGCGGAAGTAAAGAG
TCCGGCCACCGTTAAATCCCCGGTAGAGGCCAAAAGCCCTGCGGAAG
TTAAATCGCCGGTGACGGTCAAATCACCCGCGGAAGCGAAGTCCCCG
GTGGAGGTGAAATCTCCGTACTGGTGTGCCTAA
UNDERLINE UPPER CASE = Thrombin cleavage site
BOLD UPPER CASE = IDP nucleic acid
KSPAEAKSPAEVKSPAVAKSPAEVKSPAEVKSPAEAKSPAEAKSPAE
VKSPATVKSPGEAKSPAEAKSPAEVKSPVEAKSPAEAKSPASVKSPG
EAKSPAEAKSPAEVKSPATVKSPVEAKSPAEVKSPVTVKSPAEAKSP
VEVKSPYWCA (SEQ ID NO: 36)
UNDERLINE UPPER CASE = Thrombin cleavage site
BOLD UPPER CASE = IDP polypeptide
An exemplary IDP-ketosteroid isomerase protein (KSI) amino acid sequence is provided in Table 3B.
VKSPAVAKSPAEVKSPAEVKSPAEAKSPAEAKSPAEVKSPATVKSPG
EAKSPAEAKSPAEVKSPVEAKSPAEAKSPASVKSPGEAKSPAEAKSP
AEVKSPATVKSPVEAKSPAEVKSPVTVKSPAEAKSPVEVKSPYWSA
UNDERLINE UPPER CASE = CNBr cleavage site
BOLD UPPER CASE = IDP polypeptide
Exemplary sequences for amphiphilic fusion proteins of the present technology, comprising a hydrophobic polypeptide sequence fused to a hydrophilic polypeptide sequence, and designated as IDP1-2Yx2A, IDP2-2Yx2A, IDP-2Yx3A, and IDP-2Yx4A are set forth in Tables 4A, 4B, 5, and 6 respectively.
AAGAGTCCAGCGGAAGCTAAGTCGCCAGCCGAAGTCAAGTCGCCCGC
CGTCGCGAAAAGCCCCGCAGAGGTGAAATCCCCGGCCGAAGTCAAAT
CGCCGGCAGAAGCGAAATCCCCGGCAGAAGCAAAAAGTCCTGCTGAG
GTCAAATCGCCAGCAACCGTCAAATCCCCTGGAGAGGCAAAATCTCC
GGCAGAAGCCAAGTCCCCTGCCGAAGTGAAGTCACCTGTCGAAGCCA
AGTCGCCGGCCGAAGCGAAGAGCCCAGCGAGCGTGAAAAGTCCTGGT
GAGGCTAAGTCCCCGGCGGAAGCGAAATCTCCAGCGGAAGTAAAGAG
TCCGGCCACCGTTAAATCCCCGGTAGAGGCCAAAAGCCCTGCGGAAG
TTAAATCGCCGGTGACGGTCAAATCACCCGCGGAAGCGAAGTCCCCG
GTGGAGGTGAAATCTCCGTACTGGTGTGCC
TATGGCGCGTATGCGCA
GTATGTGTATATTTATGCGTATTGGTATCTGTAA
UNDERLINE UPPER CASE = Thrombin cleavage site
BOLD UPPER CASE = IDP nucleic acid
UNDERLINED BOLD UPPER CASE
= Hydrophobic
KSPAEAKSPAEVKSPAVAKSPAEVKSPAEVKSPAEAKSPAEAKSPAE
VKSPATVKSPGEAKSPAEAKSPAEVKSPVEAKSPAEAKSPASVKSPG
EAKSPAEAKSPAEVKSPATVKSPVEAKSPAEVKSPVTVKSPAEAKSP
VEVKSPYWCA
YGAYAQYVYIYAYWYL
(SEQ ID NO: 38)
UNDERLINE UPPER CASE = Thrombin cleavage site
BOLD UPPER CASE = IDP polypeptide
UNDERLINED BOLD UPPER CASE
=
AWRGSPWAEAKSPAEAKSPAEVKSPAVAKSPAEVKSPAEVKSPAEAK
SPAEAKSPAEVKSPATVKSPGEAKSPAEAKSPAEVKSPVEAKSPAEA
KSPASVKSPGEAKSPAEAKSPAEVKSPATVKSPVEAKSPAEVKSPVT
VKSPAEAKSPVEVKSPYWCA
YGAYAQYVYIYAYWYL
BOLD UPPER CASE = IDP polypeptide
UNDERLINED BOLD UPPER CASE
=
VKSPAVAKSPAEVKSPAEVKSPAEAKSPAEAKSPAEVKSPATVKSPG
EAKSPAEAKSPAEVKSPVEAKSPAEAKSPASVKSPGEAKSPAEAKSP
AEVKSPATVKSPVEAKSPAEVKSPVTVKSPAEAKSPVEVKSPYWSA
Y
GAYAQYVYIYAYWYLM
UNDERLINE UPPER CASE = CNBr cleavage site
BOLD UPPER CASE = IDP polypeptide
UNDERLINED BOLD UPPER CASE
=
= His-tag
LAEAKSPAEAKSPAEVKSPAVAKSPAEVKSPAEVKSPAEAKSPAEAK
SPAEVKSPATVKSPGEAKSPAEAKSPAEVKSPVEAKSPAEAKSPASV
KSPGEAKSPAEAKSPAEVKSPATVKSPVEAKSPAEVKSPVTVKSPAE
AKSPVEVKSPYWSA
YGAYAQYVYIYAYWYL
BOLD UPPER CASE = IDP polypeptide
UNDERLINED BOLD UPPER CASE
=
AAGAGTCCAGCGGAAGCTAAGTCGCCAGCCGAAGTCAAGTCGCCCGC
CGTCGCGAAAAGCCCCGCAGAGGTGAAATCCCCGGCCGAAGTCAAAT
CGCCGGCAGAAGCGAAATCCCCGGCAGAAGCAAAAAGTCCTGCTGAG
GTCAAATCGCCAGCAACCGTCAAATCCCCTGGAGAGGCAAAATCTCC
GGCAGAAGCCAAGTCCCCTGCCGAAGTGAAGTCACCTGTCGAAGCCA
AGTCGCCGGCCGAAGCGAAGAGCCCAGCGAGCGTGAAAAGTCCTGGT
GAGGCTAAGTCCCCGGCGGAAGCGAAATCTCCAGCGGAAGTAAAGAG
TCCGGCCACCGTTAAATCCCCGGTAGAGGCCAAAAGCCCTGCGGAAG
TTAAATCGCCGGTGACGGTCAAATCACCCGCGGAAGCGAAGTCCCCG
GTGGAGGTGAAATCTCCGTACTGGTGTGCC
TATGGCGCGTATGCGCA
GTATGTGTATATTTATGCGTATTGGTATCTGTATGCTTATATTTAA
UNDERLINE UPPER CASE = Thrombin cleavage site
BOLD UPPER CASE = IDP nucleic acid
UNDERLINED BOLD UPPER CASE
= Hydrophobic
KSPAEAKSPAEVKSPAVAKSPAEVKSPAEVKSPAEAKSPAEAKSPAE
VKSPATVKSPGEAKSPAEAKSPAEVKSPVEAKSPAEAKSPASVKSPG
EAKSPAEAKSPAEVKSPATVKSPVEAKSPAEVKSPVTVKSPAEAKSP
VEVKSPYWCA
YGAYAQYVYIYAYWYLYAYI
(SEQ ID NO: 41)
UNDERLINE UPPER CASE = Thrombin cleavage site
BOLD UPPER CASE = IDP polypeptide
UNDERLINED BOLD UPPER CASE
=
AWRGSPWAEAKSPAEAKSPAEVKSPAVAKSPAEVKSPAEVKSPAEAK
SPAEAKSPAEVKSPATVKSPGEAKSPAEAKSPAEVKSPVEAKSPAEA
KSPASVKSPGEAKSPAEAKSPAEVKSPATVKSPVEAKSPAEVKSPVT
VKSPAEAKSPVEVKSPYWCA
YGAYAQYVYIYAYWYLYAYI
BOLD UPPER CASE = IDP polypeptide
UNDERLINED BOLD UPPER CASE
=
AWRGSPWAEAKSPAEAKSPAEVKSPAVAKSPAEVKSPAEVKSPAEAK
SPAEAKSPAEVKSPATVKSPGEAKSPAEAKSPAEVKSPVEAKSPAEA
KSPASVKSPGEAKSPAEAKSPAEVKSPATVKSPVEAKSPAEVKSPVT
VKSPAEAKSPVEVKSPYWCA
YGAYAQYVYIYAYWYLYAYIAVAL
BOLD UPPER CASE = IDP polypeptide
UNDERLINED BOLD UPPER CASE
=
The present section provides a general description of the synthesis, formation and use of the amphiphilic fusion proteins and micellar compositions as described herein. Plasmids encoding the 2Yx2A, 2Yx3A, or 2Yx4A amphiphilic fusion proteins of the present technology are prepared according to the methods outlined in the Examples.
Any suitable expression system for producing the amphiphilic fusion proteins may be employed. In some embodiments, a cell-free system is used for the production of the amphiphilic fusion proteins. In some embodiments, a host cell is transformed with the expression vectors of the present technology. In some embodiments, the host cell is any eukaryotic, prokaryotic, or archaea cell. In some embodiments, the host cell is a yeast, bacterial, cyanobacteria, insect, plant, or mammalian cell. In some embodiments, the host cell is E. coli.
In some embodiments, the present disclosure relates to cell cultures comprising the host cells transformed with the expression vectors comprising chimeric nucleic acids encoding the amphiphilic fusion proteins of the present technology.
The expressed fusion proteins are then digested with a protease (e.g., thrombin) to remove the solubilizing moiety and may then be purified by any suitable means known in the art. Non-limiting purification methods are further described in the Examples.
Also provided herein in one aspect is a method of producing an amphiphilic fusion protein that spontaneously self-assembles to form a stable micelle, the method comprising: (a) introducing into a host cell an expression vector comprising a chimeric nucleic acid construct comprising, in the 5′ to 3′ direction, a promoter suitable for directing expression in a host cell operably linked to a nucleic acid sequence encoding an amphiphilic fusion protein having Formula (I): S—X—H1—H2, wherein S— is a solubilizing moiety, —X— is a peptide sequence comprising a proteolytic cleavage site, —H1— is a hydrophilic peptide, and —H2 is a hydrophobic peptide; (b) growing the host cell under conditions that allow for expression of the chimeric nucleic acid to produce the amphiphilic fusion protein; (c) purifying the amphiphilic fusion protein; and (d) contacting the amphiphilic fusion protein with a protease to provide an amphiphilic fusion protein having Formula (II): H1—H2. In some embodiments, the chimeric nucleic acid construct of part (a) encodes an amphiphilic fusion protein further comprising a cell targeting peptide (-T-) between the —X— and the —H1—, such that the amphiphilic fusion protein has Formula (III): S—X-T-H1—H2, and such that after part (d) the amphiphilic fusion protein has Formula (IV): T-H1—H2.
The expressed fusion proteins are then digested with reagent to induce chemical cleavage (e.g., CNBr) to remove the insolubilizing moiety and may then be purified by any suitable means known in the art. Non-limiting purification methods are further described in the Examples.
Also provided herein in one aspect is a method of producing an amphiphilic fusion protein that spontaneously self-assembles to form a stable micelle, the method comprising: (a) introducing into a host cell an expression vector comprising a chimeric nucleic acid construct comprising, in the 5′ to 3′ direction, a promoter suitable for directing expression in a host cell operably linked to a nucleic acid sequence encoding an amphiphilic fusion protein having Formula (I): I—X—H1—H2, wherein I— is an insolubilizing moiety, —X— is a peptide sequence comprising a chemical cleavage site, —H1— is a hydrophilic peptide, and —H2 is a hydrophobic peptide; (b) growing the host cell under conditions that allow for expression of the chimeric nucleic acid to produce the amphiphilic fusion protein; (c)purifying the amphiphilic fusion protein; and (d) contacting the amphiphilic fusion protein with a reagent to induce chemical cleavage to provide an amphiphilic fusion protein having Formula (II): H1—H2. In some embodiments, the chimeric nucleic acid construct of part (a) encodes an amphiphilic fusion protein further comprising a cell targeting peptide (-T-) between the —X— and the —H1—, such that the amphiphilic fusion protein has Formula (III): I-X-T-H1—H2, and such that after part (d) the amphiphilic fusion protein has Formula (IV): T-H1—H2.
Provided in another aspect are micelles comprising any one of the amphiphilic fusion proteins described herein. In some embodiments, the amphiphilic fusion protein is characterized by a hydrophilic peptide (H1); and a hydrophobic peptide (H2).
In some embodiments, the micelle described herein has a low critical micelle concentration (CMC). In some embodiments, the CMC of the amphiphilic fusion protein in water is greater than about 10 μM at a physiological pH of about 7.4. In some embodiments, the CMC of the amphiphilic fusion protein in water is less than about 20 μM at a physiological pH of about 7.4. In some embodiments, the CMC of the amphiphilic fusion protein in water is from about 10 μM to about 20 μM at a physiological pH of about 7.4. In some embodiments, the CMC of the amphiphilic fusion protein in water is about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 18 μM, about 19 μM, or about 20 μM at a physiological pH of about 7.4.
In some embodiments, the micelle described herein has a diameter from about 20 nm to about 40 nm. In some embodiments, the micelle described herein has a diameter greater than about 20 nm. In some embodiments, the micelle described herein has a diameter less than about 40 nm. In some embodiments, the micelle described herein has a diameter of about 20 nm, about 21 nm, about 22 nm, about 23 nm, about 24 nm, about 25 nm, about 26 nm, about 27 nm, about 28 nm, about 29 nm, about 30 nm, about 31 nm, about 32 nm, about 33 nm, about 34 nm, about 35 nm, about 36 nm, about 37 nm, about 38 nm, about 39 nm, or about 40 nm. In some embodiments, the micelle described herein has a diameter of about 27 nm.
In some embodiments, the micelle described herein is pH stable. In some embodiments, the micelle is stable at a pH from about 2.0 to about 10.0. In some embodiments, the micelle is stable at a pH greater than about 2.0. In some embodiments, the micelle is stable at a pH less than about 10.0. In some embodiments, the micelle is stable at a pH of about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, or to about 10.0.
In some embodiments, the micelle described herein is temperature stable. In some embodiments, the micelle is stable at a temperature from about 25° C. to about 70° C. In some embodiments, the micelle is stable at a temperature greater than about 25° C. In some embodiments, the micelle is stable at a temperature less than about 70° C. In some embodiments, the micelle is stable at a temperature of about 25° C., about 30° C., about 35° C., about 40° C., about 45° C., about 50° C., about 55° C., about 60° C., about 65° C., about 70° C., or about 75° C.
In some embodiments, the micelle further contains a fluorescent dye. In some embodiments, the fluorescent dye is covalently attached to the hydrophilic peptide (H1). In some embodiments, the fluorescent dye is covalently attached to the hydrophobic peptide (H2). In some embodiments, the fluorescent dye is fluorescein or rhodamine.
In some embodiments, the micelle has a core-shell structure. In some embodiments, the micelle has a shell diameter from about 40 nm to about 75 nm. In some embodiments, the micelle has a shell diameter greater than about 40 nm. In some embodiments, the micelle has a shell diameter less than about 75 nm. In some embodiments, the micelle has a shell diameter of about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, or about 75 nm. In some embodiments, the micelle has a core diameter from about 25 nm to about 45 nm. In some embodiments, the micelle has a core diameter greater than about 25 nm. In some embodiments, the micelle has a core diameter less than about 45 nm. In some embodiments, the micelle has a core diameter of about 25 nm, about 30 nm, about 35 nm, about 40, or about 45 nm. In some embodiments, the micelle has a shell thickness from about 5 nm to about 20 nm. In some embodiments, the micelle has a shell thickness of greater than about 5 nm. In some embodiments, the micelle has a shell thickness of less than about 20 nm. In some embodiments, the micelle has a shell thickness of about 5 nm, about 10 nm, about 15 nm, or about 20 nm.
In some embodiments, the micelle further contains a hydrophobic cargo. In some embodiments, the hydrophobic cargo is a drug, a fungicide, a protein, a nucleic acid, a hormone, a receptor, a diagnostic agent, an imaging agent, a metal complex, a silicone oil, a triglyceride, or a combination thereof. In some embodiments, the hydrophobic cargo is a drug. In some embodiments, the hydrophobic cargo is a fungicide. In some embodiments, the hydrophobic cargo is a protein. In some embodiments, the hydrophobic cargo is a nucleic acid. In some embodiments, the hydrophobic cargo is a hormone. In some embodiments, the hydrophobic cargo is a receptor. In some embodiments, the hydrophobic cargo is a diagnostic agent. In some embodiments, the hydrophobic cargo is an imaging agent. In some embodiments, the hydrophobic cargo is a metal complex. In some embodiments, the hydrophobic cargo is a silicone oil. In some embodiments, the hydrophobic cargo is a triglyceride.
In another aspect, compositions, e.g., “pharmaceutical compositions” are provided comprising and an effective amount of a micelle described herein, a hydrophobic cargo, and/or a therapeutically active agent. In some embodiments, the composition further includes at least one pharmaceutically acceptable excipient.
Pharmaceutical compositions comprising a micelle described herein, a hydrophobic cargo, and/or a therapeutically active agent can be formulated for different routes of administration, including intravenous, intraarterial, pulmonary, rectal, nasal, vaginal, lingual, intramuscular, intraperitoneal, intracutaneous, transdermal, intracranial, subcutaneous and oral routes. Other dosage forms include tablets, capsules, pills, powders, aerosols, suppositories, parenterals, and oral liquids, including suspensions, solutions and emulsions. All dosage forms may be prepared using methods that are standard in the art (see e.g., Remington's Pharmaceutical Sciences, 16th ed., A. Oslo editor, Easton Pa. 1980).
In some embodiments, the micelle described herein, hydrophobic cargo, and/or therapeutically active agent are formulated in conjunction with appropriate salts and buffers to render delivery of the compositions in a stable manner to allow for uptake by target cells. Buffers also are employed when the micelle described herein, hydrophobic cargo, and/or therapeutically active agent are introduced into a patient. In some embodiments, an aqueous composition is used, comprising an effective amount of the micelle, hydrophobic cargo, and/or therapeutically active agent, which are dispersed in a pharmaceutically acceptable carrier or excipient an aqueous medium. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. Sterile phosphate-buffered saline is one example of a pharmaceutically suitable excipient. Other suitable carriers and excipients are well-known to those in the art, see, for example, Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company 1990), and revised editions thereof.
The micelle as described herein, hydrophobic cargo, and/or therapeutically active agent may be administered parenterally or intraperitoneally or intratumorally. Solutions of the active compounds as free base or pharmacologically acceptable salts are prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
Also provided is an effective method of using the amphiphilic fusion proteins and micellar compositions described herein for delivering hydrophobic cargo and/or therapeutically active agents to the interior of target cells (e.g., cancer cells, fungal cells, microbial cells). Thus, in some embodiments, methods of therapy are provided that comprise or require delivery of hydrophobic cargo and/or therapeutically active agents into a cell. In some embodiments, the hydrophobic cargo and/or therapeutically active agent is a chemotherapeutic drug, e.g., doxorubicin.
In another aspect, a method for treating cancer in a subject is provided, where the method comprises administering to the subject an effective amount of a composition comprising any of the micelles described herein and a therapeutically active agent (e.g., a chemotherapeutic drug). In some embodiments, examples of chemotherapeutic drugs include, but are not limited to, doxorubicin, paclitaxel, and rapamycin. In some embodiments, the therapeutically active agent is a steroidal drug, including, but not limited to, hydrocortisone, testosterone, progesterone, 17β-estradiol, or levonorgestrel. In some embodiments, the micelles of the present technology may be used in methods for delivering to target cells or tissues drugs that are otherwise encapsulated in polymer nanoparticles for efficient delivery including, but not limited to, risperidone, minocycline hydrochloride, or bromocriptine. In some embodiments, the micelles of the present technology are useful in methods for delivering to target cells or tissues imaging agents including, but not limited to, fluorescent dyes, PET probes, and MRI contrast agents.
The dosage of an administered micelle described herein, and hydrophobic cargo, and/or a therapeutically active agent for humans will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition and previous medical history.
In some embodiments, methods and compositions are provided for the treatment of cancer. Cell proliferative disorders, or cancers, contemplated to be treatable with the methods include, but are not limited to, human head and neck solid tumors, breast carcinoma, prostate carcinoma, hepatocellular carcinoma, adenocarcinomas. In some embodiments, the method is used to inhibit growth, progression, and/or metastasis of cancers, in particular those listed above.
The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially the same or similar results. The examples should in no way be construed as limiting the scope of the present technology, as defined by the appended claims.
This example describes the preparation of exemplary fusion proteins described herein.
Design of Sequences. The intrinsically disordered sequence derived from the neurofilament heavy arm side chain is a naturally stimulus responsive sequence that flares out around the head domain, giving rise to a cylindrical brush structure. Due to the interesting properties of this highly charged and repetitive sequence, methods were developed to express this portion of the protein with increasing hydrophobic appendages in attempt to create an intrinsically disordered protein that can self-assemble around a genetically encoded hydrophobic sequence (
Two main challenges arise with recombinantly expressed proteins such as these:
To address these issues, the constructs were expressed with an N-terminal Maltose Binding Protein (MBP) and a 6×His tag that could then be cleaved from the protein of interest by inserting a thrombin cleavage site between the two proteins. MBP serves to enhance the solubility of the protein constructs during expression and initial steps of purification, allowing for normal protein expression handling techniques. MBP also increases expression yields of constructs it is appended to which is also beneficial for production purposes.
Construction of Plasmids. (a) IDP-MBP plasmid: The inherent repetitive sequence of IDP made the synthesis of one contiguous gene block impossible. Instead, two gene blocks (gBlocks: IDT Technologies) had to be synthesized (IDP-1 and IDP2; see below for respective sequences) with a 32 bp consensus sequence that allows for Gibson assembly. A 100 ng sample of each gBlock and 10 μl of 2× Gibson master mix (ref. Gibson, D. G., Young, L., et al. Nat. Methods. 2009, 6, 343-345) was adjusted with water to a volume of 20 μL and was incubated at 50° C. for 60 min. After DNA clean up with QiaQuick (Qiagen), the assembly product was PCR amplified (VENT polymerase from NEB, Tm=61° C.) with forward and reverse primers: 5′-ATA ATA GCT AGC TTA GTT CCT CGT GCC TGG CGT G-3′ (SEQ ID NO: 43) and 5′-TAT TAT CTC GAG CTA TTA GGC ACA CCA GTA CGG AGA TTT C-3′ (SEQ ID NO: 44). The IDT insert contained NheI and XhoI restriction sites, that were double digested, heat inactivated at 80° C. for 5 min and ligated (QuickLigase, NEB) with a 5′-terminal MBP pSKB3 vector. Plating on Kanamycin agar plates yielded individual colonies that were cultured, DNA purified (NucleoSpin, MacheryNagel) and sequenced (Quintara BioSciences).
AGAAGCCAAGTCCCCTGCCGAAGTGAAGTCACCTGTCGAAGCCAAGT
(b) 2Y-MBP plasmid: Overhang PCR was performed on the MBP-IDP plasmid constructed in (a). The forward primer extended the sequence with a Bsa1 cut site while the reverse primer extended the sequence with the desired hydrophobic portion and a Bsa1 cut site to allow for incorporation into our plasmid by golden gate assembly. The amplified sequence was run on a 1% agarose gel and confirmed to be of the approximate length. The PCR product was extracted and purified. To perform the golden gate assembly, the 2Y PCR product was incubated with our golden gate plasmid, Bsa1, NEB ligase buffer, and ligase enzyme and cycled 25 times. After ligation plasmids were transformed into chemically competent cells and plated on Kanamycin LB agar plates at 37° C. overnight. When the agar plate was exposed to UV light, white colonies were selected (green indicating no excision of GFP by Bsa1) and grown in 10 mL of LB media at 37° C. overnight. Plasmid DNA was subsequently purified (NucleoSpin, MacheryNagel) and sequenced (Quintara BioSciences).
(c) 2Yx2A plasmid construction: Overhang PCR was performed on 2Y-MBP plasmid constructed in (b). The forward primer extended the sequence with a Bsa1 cut site while the reverse primer extended the sequence with a hydrophobic portion and a Bsa1 cut site to allow for incorporation into our plasmid by golden gate assembly. Following the same procedure as in (b) the 2Yx2A plasmid was purified and sequenced.
(d) 2Yx3A plasmid construction: Overhang PCR containing was performed on 2Yx2A plasmid constructed in (c). The forward primer extended the sequence with a Bsa1 cut site while the reverse primer extended the sequence with a hydrophobic portion and a Bsa1 cut site to allow for incorporation into our plasmid by golden gate assembly. Following the same procedure as in (c) the 2Yx3A plasmid was purified and sequenced.
Expression of MBP-IDP. (a) Plasmids were transformed into E. coli BL21 (DE3) competent cells. Starter cultures (20 ml LB, 50 mg/L Kanamycin) were grown from single colonies, grown overnight at 37° C., and used to inoculate 1 L of TB media (50 mg/L Kanamycin). Cultures were grown to an OD ˜0.5, cooled for 20 min at 25° C., induced with 0.5 mM IPTG, and expressed overnight (˜18 hours) at 25° C. Cells were harvested by centrifugation for 15 min at 4,000 rcf (g) at 4° C.
Purification of MBP-IDP fusion protein. The pellet was transferred to a 50 ml Falcon tube in PBS buffer, and spun down for 10 min at 4000 rcf (g). The resulting pellet (˜5 g) was lysed in 30 ml of lysis buffer (20 mM HEPES, pH=7.5, 300 mM NaCl, 10 mM imidazole=buffer A) supplemented with one tablet of EDTA-free SigmaFast Protease Inhibitor (Sigma Aldrich), 2 mM PMSF, and 10 mg lysozyme. The resuspended sample was lysed with an Avestin C3 homogenizer followed by 20 min of centrifugation at 24,000 rcf (g) at 4° C. The supernatant was filtered through a 40 μm Steriflip filter (Millipore), and loaded onto a 5 ml NiNTA column (Protino, Machery Nagel) connected to an Akta purifier that was pre-equilibrated with buffer A. The column was washed with 50 ml (10 CV) of 20 mM HEPES (pH=7.5), 300 mM NaCl, 10 mM imidazole, 10 mM βMe. The protein was eluted with 20 mM HEPES (pH=7.5), 300 mM NaCl, 250 mM imidazole, 10 mM βMe. Imidazole was removed by exchanging against 20 mM HEPES (pH=7.5), 100 mM NaCl with a 10DG desalting column (BioRad), and subsequently digested with 1 mg of thrombin protease (high purity from Bovine, MP Biomedicals). Complete digestion was achieved at room temperature after 1 hour as confirmed by LC/MS (ESI/TOF). The protein mixture was diluted with salt-free HEPES buffer (20 mM, pH=7.5) to 50 ml ([NaCl]˜5 mM) before loading it onto a 1 ml HiTrap Q HP cation exchange column connected to an Akta purifier (GE Healthcare). The column was pre-equilibrated with 20 mM HEPES, 10 mM βMe, washed with 10 ml (10 CV) of 20 mM HEPES (pH=7.5), 10 mM βMe and eluted with a gradient from 0-1M NaCl (50 ml total volume). Monomeric IDP eluted at around 200 mM NaCl. Without addition of reducing agent, significant dimeric formation that elutes with little retention at the tail end of the loading step was observed. The protein was eluted with 20 mM HEPES (pH=7.5), 300 mM NaCl, 250 mM imidazole, 10 mM βMe. The final IDP sample was obtained with a final desalting column (10DG, BioRad) to obtain the protein at a final concentration of 270 μM in 20 mM HEPES (pH=7.5), 50 mM NaCl. The protein was >95% pure by SDS-PAGE and LCMS (ESI-TOF; Agilent). The purified protein was flash frozen with liquid N2 in 20 μl aliquots.
Expression and Purification of 2Yx2A-MBP. (b) Expression: Plasmids were transformed into E. coli Rosetta 2 plys competent cells. Starter cultures (20 ml LB, 50 mg/L Kanamycin) were grown from single colonies, grown overnight at 37° C., and used to inoculate 1 L of TB media (50 mg/L Kanamycin). Cultures were grown to an OD ˜0.5, cooled for 20 min at 16° C., induced with 0.1 mM IPTG, and expressed (˜6 hours) at 16° C. Cells were harvested by centrifugation for 15 min at 4,000 rcf (g) at 4° C.
(c) Purification of MBP-2Yx2A fusion protein: The pellet was transferred to a 50 ml Falcon tube in PBS buffer, and spun down for 10 min at 4000 rcf (g). The resulting pellet (˜5 g) was lysed in 30 ml of lysis buffer (20 mM HEPES, pH=7.5, 300 mM NaCl, 10 mM imidazole=buffer A) supplemented with one tablet of EDTA-free SigmaFast Protease Inhibitor (Sigma Aldrich), 2 mM PMSF, and 10 mg lysozyme. The resuspended sample was lysed by sonication (amplitude 50%, 2:4 seconds on off for 10 minutes) followed by 20 min of centrifugation at 24,000 rcf (g) at 4° C. The supernatant was filtered through a 40 μm Steriflip filter (Millipore), and loaded onto a 5 ml NiNTA column (Protino, Machery Nagel) connected to an Akta purifier that was pre-equilibrated with buffer A. The column was washed with 50 ml (10 CV) of 20 mM HEPES (pH=7.5), 300 mM NaCl, 10 mM imidazole. The protein was eluted with 20 mM HEPES (pH=7.5), 300 mM NaCl, 250 mM imidazole. Imidazole was removed by spin concentration with 20 mM HEPES (pH=7.5), 100 mM NaCl. MBP-2Yx2/3A was subsequently digested with 1 mg of thrombin protease (high purity from Bovine, MP Biomedicals). Complete digestion was achieved at room temperature after 1 hour as confirmed by LC/MS. Then either ion exchange or Biotage HPLC purification could be used to remove the residual MBP.
(d) Ion exchange: The protein mixture was diluted with salt-free HEPES buffer (20 mM, pH=7.5) and 8M urea to 50 ml ([NaCl]˜5 mM) before loading it onto a 1 ml HiTrap SP HP cation exchange column connected to an Akta purifier (GE Healthcare). The column was pre-equilibrated with 20 mM HEPES, 10 mM βMe, washed with 10 ml (10 CV) of 20 mM HEPES (pH=7.5) and eluted with a gradient from 0-1 M NaCl (100 ml total volume). 2Yx2A eluted at around 500 mM NaCl. The final 2Yx2A sample was dialyzed against 100 mM PB pH 5.5. Gel analysis shows 75% purity.
(e) Biotage HPLC: 10% acetonitrile (ACN) is added to the crude protein mixture which is then loaded onto a 10 g C18 Biotage SNAP Bio 300A reversed phase column that has been equilibrated with 10% ACN in H2O+0.1% TFA. The column was run over 14 minutes to 100% ACN with the desired product eluting around 40% ACN. The fractions containing 2Yx2A were analyzed by LC/MS (ESI/TOF) for purity. 100% pure fractions were collected and lyophilized to dryness resulting in a white powder. Gel analysis shows >95% purity.
The purity and characterization of the 2Yx2A-MBP proteins by gel and LC/MS are shown in
Expression and Purification of 2Yx3A-MBP. (f) Expression: 2Yx3A-MBP was expressed in the same way as 2Yx2A-MBP (b).
(g) Purification: 2Yx3A-MBP was purified in the same was as 2Yx2A-MBP (f).
In contrast to the expression of IDP-MBP and 2Yx2A-MBP, 2Yx3A-MBP resulted in low pellet volume during expressing. Additionally, after cell lysis by sonication, the protein supernatant resembled sudsy soap making it difficult to handle and hard to purify. The surfactant like nature of 2Yx3A-MBP was still present after NiNTA purification something not seen with the 2Yx2A-MBP construct. This is interesting because the only difference in the two sequences is the addition of four amino acids (YAYI) to the 2Yx3A-MBP sequence.
The characterization of the 2Yx3A-MBP proteins are shown in
This example provides characterization data of the micelles prepared from the exemplary fusion proteins described herein.
The resulting micelle from the 2Yx2A construct was characterized fully and in some cases the non-assembling IDP construct was used for comparison. The micelle from the 2Yx3A construct was not further characterized due to difficulties with expression and purification of the 2Yx3A construct. Briefly, to analyze the 2Yx2A, the lyophilized protein was resuspended in water and then adjusted to the desired buffer conditions.
Dynamic Light Scattering. DLS analysis was conducted on a Malvern Instruments Zetasizer Nano ZS. Data plots and standard deviations are calculated from an average of three measurements, each of which consisted of 13 runs. Measurement data is presented as a diameter determined by the % Number distribution. When measuring any of the assembling constructs, the protein module could be used to analyze the particles, however, to obtain any signal for IDP it must be treated as a polymer and diluted to low concentrations.
Size Exclusion Chromatography.
Critical Micelle Concentration Determination by Pyrene Fluorescence. The CMC of 2Yx2A and IDP in 100 mM PB pH 5.8 was analyzed by measuring the first and third vibronic band of pyrene (I1/I3-ratio) which increases with increasing polarity of the probe environment. For example, the I1/I3 ratio in water is 1.32 while in cyclohexane is it 0.6.
To each sample, 2 μM of pyrene was added and let equilibrate for 5 minutes. Each protein solution was then diluted with a solution of 2 μM pyrene in the buffer to keep pyrene and salt concentration constant, but decrease protein concentration. The emission spectrum of pyrene was collected on a Horiba fluorimeter exciting at 335 nm with a 5 nm window and monitoring emission from 350-800 nm. At higher protein concentrations a peak at 383 nm corresponding to pyrenes third vibronic band is present, while at lower concentrations the intensity of this band decreases (
When the I1/I3 ratio is plotted against 2Yx2A protein concentration, a Boltzmann fit can be applied to the data points to calculate an EC50 value. In 1×PBS pH 7.4 this value is 26 μM while in 100 mM PB pH 5.7 this value is 13 μM. Alternatively, when this same analysis is applied to IDP, the I1/I3 ratio remains constant and exhibits that of water (0 uM protein) overall concentrations up to 100 μM. The I1/I3 ratio of pyrene in high concentrations (above 30 μM) of 2Yx2A closely resembles that of pyrene in 1-propanol ˜1.0 (
Micelle exchange dynamics measured using FRET. (a) Labeling of internal cysteine with fluorescent dye: To measure the exchange dynamics of the 2Yx2A micelles, two separate populations of 2Yx2A were labeled with either Fluorescein maleimide or Rhodamine red maleimide on the internal cysteine residues that sits at between the hydrophobic and hydrophilic portion of the protein. After 24 h, both populations showed 4% labeling (
To remove excess dye, the proteins were purified using a NAPS column resulting in 600 μL of protein at 1 μM (below the CMC). To this solution, 50 μL of 40 uM 2Yx2A was added and then spin concentrated with a 3 KDa MWCO. This should achieve approximately 1% labeling of all 2Yx2A protein monomers. Assuming aggregation numbers in the hundreds this correlates to a low average number of dye molecules per micelle.
(b) FRET analysis: 2Yx2A-FITC and 2Yx2A-RhoRED were individually excited using a Horiba fluorometer with 490 nm light 5 nm window and the emission spectrum was collected from 500-800 nm in 1 nm increments. Then 30 μL of each solution was mixed together and then immediately analyzed. Consecutive time points over 40 hs were then taken.
Cryo TEM. Cryo TEM samples were prepared from a 12 μM stock solution of 2Yx2A in 100 mM PB pH 5.3 before analysis samples were diluted 30-fold to a concentration of 0.4 μM for analysis. By DLS the average diameter and standard deviation observed for these particles was slightly larger due to the dilution (see
SAXS. This work benefited use of the SasView application(M. Doucet, et al. SasView Version 4.1.2).
This example is illustrative in demonstrating that the micelles prepared from the exemplary fusion proteins described herein are capable of being loaded with a hydrophobic compound, such as pyraclostrobin.
Pyraclostrobin is a highly water-insoluble organic compound that is also a potent fungicide. Its solubility in water is reported to be 1.9 mg/L. As a control a saturated solution of pyraclostrobin was created by adding it to 100 mM PB pH 5.3 and measuring its absorbance at 280 nm, as expected there was no observable signal. To determine if pyraclostrobin could be taken up into 2Yx2A micelles, pyraclostrobin was added until it saturated a solution of 100 μL of 11 μM (A280: 0.361) 2Yx2A, evidenced by yellow particulates. The solution was then centrifuged and the supernatant was removed and then placed in a fresh tube and centrifuged again. Supernatant removal and centrifugation were repeated 3 times to remove any insoluble pyraclostrobin that has not partitioned into the micelle interior. The solution was then measured with a nanodrop to determine its absorbance at 280 nm resulting in an A280 of 0.502 (+0.141 mAU). Given the pyrene extinction coefficient of 24,000 at A275, this increase in absorbance corresponds to the presence of 5.8 μM pyrene. Similarly, the same sample can be analyzed for pyraclostrobin content via HPLC using a calibration curved developed using known pyrene concentrations in acetonitrile (
Instead of directly adding pyraclostrobin to a solution of 2Yx2A micelles an alternative method was used to achieve higher loading efficiency. This method involved the co-resuspension of lyophilized protein and pyraclostrobin in THF which is then slowly diluted with buffer. The solution was then centrifuged and the supernatant was removed and then placed in a fresh tube and centrifuged again. Supernatant removal and centrifugation were repeated 3 times to remove any insoluble pyraclostrobin that has not partitioned into the micelle interior. The sample was then lyophilized to remove all solvent and then resuspended in milliQ water. Although no visible precipitation was observed, the sample was centrifuged to remove any unincorporated pyraclostrobin. For HPLC analysis the protein-pyraclostrobin solution is diluted to ½ of its volume by pyrene to break apart the micelles. Then a known volume is injected onto the HPLC. Based on the area of the pyrene peak and volume injected the number of moles and thus the concentration of pyraclostrobin can be determined.
Accordingly, these results demonstrate that the micelles of the present technology are useful in compositions for solubilizing highly water-insoluble organic compounds and can be useful in methods for delivery of such compounds to an intended target. For example, micellar compositions comprising pyraclostrobin may be useful as a fungicide.
This example is illustrative in demonstrating that the micelles prepared from the exemplary fusion proteins described herein are capable of being loaded with a hydrophobic metal complex.
To visualize the ability of the 2Yx2A micelles to load hydrophobic compounds in their core, 40 μM 2Yx2A protein was incubated with the commonly used Suzuki coupling catalyst Pd(dppf)Cl2 for 1 week at 4° C. Prior to use, the sample was centrifuged and then the supernatant was passed through a 2 μm spin filter to remove any insoluble catalyst that has not partitioned into the micelle interior. The sample was then placed onto formvar-coated carbon grids that had been hydrophilized and let sit for 2 minutes. Excess liquid was wicked away using the tip of filter paper. The grids were then dried completely prior to analysis by a TECANI 12 TEM (UC Berkeley Electron Microscopy Facility). Due to the high electrons density of the palladium and iron-containing ferrocene ligands, the contrast in the TEM images can be achieved without the use of stain (
This example describes another method for preparing the exemplary fusion proteins described herein.
This example describes an expression system where the solubilizing fusion protein, maltose binding protein (MBP), has been replaced with the inclusion body directing fusion protein ketosteroid isomerase (KSI). By installing KSI at the N-terminus of the IDP-2Yx2A protein sequence, the entire fusion protein was sent to insoluble inclusion bodies during expression. Due to the insoluble nature of the fusion protein, the fusion protein was easily purified by centrifugation after cell lysis. Use of the KSI moiety reduced the amount of solvent needed in the initial purification step and also avoided stability issues encountered with soluble proteins. To remove the KSI fusion protein from the IDP-2Yx2A sequence, a methionine (Met) residue was installed between the two protein domains (KSI-Met-IDP-2Yx2A). Upon exposure to cyanogen bromide (CNBr), the peptide bond at the C-terminus of the Met residue was hydrolyzed leaving a C-terminal lactone on the KSI fusion protein and the desired IDP-2Yx2A. The IDP-2Yx2A protein was then purified using reversed phase chromatography.
The following materials were used:
Pet31b-KSI-IDP-2Yx2A Plasmid Construction
Entry vector: The pet31b KSI entry vector was purchased from Millipore as 69952 Sigma-AldrichpET-31b(+) DNA—Novagen.
Template DNA: The MBP-IDP-2Yx2A pet28b vector was used as template DNA and overhang PCR was performed to amplify the IDP-2Yx2A sequence.
Overhang PCR primers: These were purchased from Integrated DNA Technologies.
As shown below in Table 8, overhang PCR was performed on a BioRad S1000 Thermal Cycler with an annealing temperature of 55° C. Following heat inactivation of the Phusion enzyme, 6 μL of 6× loading dye was added to each overhang PCR reaction, which was then loaded onto a 1% agarose gel pre-stained with SYBR Safe (ThermoFisher). Gel electrophoresis was performed at 120 V for 35 minutes and then imaged under UV fluorescence with a BioRad GelDoc EZ Imager. The DNA bands corresponding to approximately 500 base pairs were excised from the gel and both were placed in a 1.5 mL Eppendorf tube. The amplified PCR product was removed from the gel using the Quick Gel Extraction Kit (Promega). The amplified PCR product was eluted in a final volume of 50 μL with a concentration of 23.7 ng/μL.
The amplified PCR product and the pet31b entry vector were then digested with XhoI and AlwnI restriction enzymes to create complementary sticky ends. The parameters for the restriction digest are shown in Table 9.
The restriction digests were incubated for 1 h at 37° C. and then heat inactivated at 80° C. for 20 min. Following digestion, 10 μL of 6× loading dye was added to each sample, which was then loaded onto a 1× agarose gel pre-stained with SYBR Safe (ThermoFisher). Gel electrophoresis was performed at 120 V for 35 minutes and then imaged under UV fluorescence with BioRad GelDoc EZ Imager. Fluorescent bands corresponding to approximately 500 base pairs (insert) and 5000 base pairs (vector) were excised from the gel and purified using the quick gel extraction kit (Promega). After elution, the concentrations of the cut vector and insert DNA was 15.1 ng/μL and 13.9 ng/μL respectively.
The ligation reaction between the digested pet31b (vector) and digested amplified PCR product (insert) was performed with varying ratios of vector: insert, where the vector concentration was kept constant at 5 ng/μL. The parameters for the ligation of the digested pet31B vector is shown in Table 10.
The ligation reactions were incubated at 16° C. for 16 h and heat inactivated at 80° C. for 20 min. Half of each ligation reaction (10 μL) was transferred to separate 1.5 mL Eppendorf tubes containing 50 μL of frozen XL1 blue chemically competent cells. The ligation reactions were gently flicked to ensure proper mixing and then incubated on ice for 30 min. The transformation was performed by heat shocking the cells-ligation mixture in a 42° C. water bath for 42 secs and then put back on ice. Under sterile conditions, 950 μL of SOC media was immediately added to the Eppendorf tube, which was then placed in a 37° C. incubator with an orbital rotator set to 200 rpm. After 1 h, 200 μL of each of the four transformations were plated on separate carbenicillin agar plates, labeled with their respective insert: vector ratios, and placed in a 37° C. incubator overnight.
After overnight incubation, the transformations corresponding to the 1:3 and 1:7 ligations had the most colonies with the 1:0 negative control had fewer than 5 colonies suggesting a low background of the vector with unincorporated insert. Four colonies from each plate were scraped with a sterile pipette tip and transferred to 5 mL of LB media+carbenicillin and grow overnight in a 37° C. incubator with an orbital rotator set to 200 rpm. The overnight cultures were transferred to 1.5 mL Eppendorf tubes and centrifuged at 13.1 g for 2 min to pellet the cells. The supernatant was removed and the plasmids were extracted from the pelleted cells using a QIAGEN miniprep kit. The eluted plasmids were sent for sequencing using a T7 forward primer. Of the eight plasmids sent for sequencing, all contained the IDP-2Yx2A insert at the C-terminus of the KSI fusion protein as shown below.
VKSPAVAKSPAEVKSPAEVKSPAEAKSPAEAKSPAEVKSPATVKSPG
EAKSPAEAKSPAEVKSPVEAKSPAEAKSPASVKSPGEAKSPAEAKSP
AEVKSPATVKSPVEAKSPAEVKSPVTVKSPAEAKSPVEVKSPYWSA
Y
GAYAQYVYIYAYWYLM
;
UNDERLINE UPPER CASE = CNBr cleavage site
BOLD UPPER CASE = IDP polypeptide
UNDERLINED BOLD UPPER CASE
= Hydrophobic
= His-tag
Expression of KSI-IDP-2Yx2A. The pet31b plasmid containing KSI-IDP-2Yx2A was transformed into Rosetta2plys cells for expression by adding 1 μL of the plasmid to 50 μL Rosetta2plys cells in a 1.5 mL Eppendorf tube on ice. The cells were then gently flicked to ensure proper mixing and incubated on ice for 30 mins before heat shocking the cells in a 42° C. water bath for 42 secs. Under sterile conditions, 950 μL of SOC media was immediately added to the Eppendorf tube containing transformation, which was then placed in a 37° C. incubator with an orbital rotator set to 200 rpm. After 1 h, 200 μL of the transformation was plated on carbenicillin+chloramphenicol agar plates and placed in a 37° C. incubator overnight.
After overnight incubation, a single colony from the agar plate was picked using a sterile pipette and added to 15 mL of LB media with carbenicillin+chloramphenicol and incubated overnight. The following day the entire overnight culture was added to 1 L of sterile TB media with carbenicillin+chloramphenicol in a 4 L flask. The flask was then placed in a 37° C. incubator rotating at 200 rpm. When the optical density at 600 nm reached 0.7 the culture was cooled down to 18° C. for 30 minutes. To induce expression, 0.5 mM IPTG was added to the media and the culture was shaken at 200 rpm for an additional 18 h overnight.
After overnight expression, the culture was centrifuged at 4000 rpm for 15 minutes. The supernatant discarded, and the remaining cell pellet was split evenly and transferred to two 50 mL falcon tubes resulting in a total cell pellet weight of 10 g (5 g per falcon tube). The cell pellets were then frozen at −20° C. overnight.
Purification of KSI-IDP-2Yx2A. The frozen 5 g cell pellet was resuspended in 30 mL of lysis buffer (20 mM HEPES, 300 mM NaCl, 10 mM BMe, 0.1% Triton-X) plus 300 μL PMSF immediately before use. The cells were lysed by sonication on the ice at 70% amplitude for 30 minutes (2 secs on 4 secs off). The lysed cells were then centrifuged at 14000 rpm for 20 minutes. The supernatant was removed and discarded. The pellets were then resuspended in lysis buffer and centrifuged at 14000 rpm again. The supernatant was discarded and the pellets were then resuspended and centrifuged two more times in MilliQ H2O. The resulting pellet was then resuspended in 10 mL of 6M Guanidinium HCl and centrifuged at 14000 rpm. The desired KSI-IDP-2Yx2A protein now resided in the supernatant at which is removed from the pelleted cell debris and stored at 4 C. The protein concentration obtained in the insoluble fraction from one 5 g cell pellet (500 mL of cell culture) is approximately 180 mg by UV absorbance at 280 nm, where the approximation that an A280 of 1.0=1 mg/mL. Of the 180 mg of protein obtained, the centrifugation purification process produced a protein solution which was predominately KSI-IDP-2Yx2A when analyzed by SDS PAGE gel and LCMS (
CNBr cleavage of KSI-IDP-2Yx2A. To the 10 mL solution of KSI-IDP-2Yx2A in 6M Guanidinium HCl, 2 mL of 3M HCl and one scoop (˜50 mg) of cyanogen bromide (CNBr) was added. The solution container was wrapped in foil and stirred under nitrogen overnight. The following day complete cleavage was observed by LCMS as no mass corresponding to of the original KSI-IDP-2Yx2A was detected and the mass of IDP-2Yx2A was also observed (
HPLC Purification of IDP-2Yx2A. The IDP-2Yx2A was then then purified using reversed phase chromatography as previously described herein.
While certain embodiments have been illustrated and described, a person with ordinary skill in the art, after reading the foregoing specification, can effect changes, substitutions of equivalents and other types of alterations to the protein fusions and micelles of the present technology or derivatives, or pharmaceutical compositions thereof as set forth herein. Each aspect and embodiment described above can also have included or incorporated therewith such variations or aspects as disclosed in regard to any or all of the other aspects and embodiments.
The present technology is also not to be limited in terms of the particular aspects described herein, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. It is to be understood that this present technology is not limited to particular methods, reagents, compounds, compositions, labeled compounds or biological systems, which can, of course, vary. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting. Thus, it is intended that the specification be considered as exemplary only with the breadth, scope and spirit of the present technology indicated only by the appended claims, definitions therein and any equivalents thereof. No language in the specification should be construed as indicating any non-claimed element as essential.
The embodiments, illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” “containing,” etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the claimed technology. Additionally, the phrase “consisting essentially of” will be understood to include those elements specifically recited and those additional elements that do not materially affect the basic and novel characteristics of the claimed technology. The phrase “consisting of” excludes any element not specified.
In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the present technology. This includes the generic description of the present technology with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member, and each separate value is incorporated into the specification as if it were individually recited herein.
All publications, patent applications, issued patents, and other documents (for example, journals, articles and/or textbooks) referred to in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.
Other embodiments are set forth in the following claims, along with the full scope of equivalents to which such claims are entitled.
The present application claims the benefit of priority to U.S. Application No. 62/695,474, filed on Jul. 9, 2018, the contents of which are incorporated herein in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/040775 | 7/8/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62695474 | Jul 2018 | US |
