Patent Application
20230086188
This application is filed with a Computer Readable Form of a Sequence Listing in accord with 37 C.F.R. § 1.821(c). The text file submitted by EFS, “028193-9340-WO01_sequence_listing_2-MAR-2021_ST25.txt,” was created on Mar. 2, 2021, contains 317 sequences, has a file size of 527 Kbytes, and is hereby incorporated by reference in its entirety.
Described herein are peptide biopolymers that exhibit controlled phase separation based on their amino acid sequence, aromatic:aliphatic ratio, hydrophobicity, temperature, molecular weight, and concentration.
Intrinsically disordered proteins (IDPs) are receiving significant recognition for their role in various biological (dys)functions. A subset of IDPs, termed biological condensates, physically separate themselves from the cytoplasm to control the accessibility of a variety of macromolecules. While our ability to detect protein disorder has advanced rapidly thanks to sophisticated statistical methods, the ability to predict phase separation has lagged behind. The prediction of phase separation is non-trivial, as numerous variables influence phase separation. Broadly, they involve: (1) amino acid composition and amino acid patterning of the primary protein sequence; (2) heterotypic interactions with RNA or other macromolecules; and (3) solvent quality. There are many studies that note the challenge of predicting IDP phase behavior, but few studies that have directly tackled this problem. Given the recognition of its importance to cellular function, this is now an area of active research and many efforts are ongoing using computational and experimental approaches. To date, however, most experimental methods to develop a sequence level understanding of IDP phase behavior have relied on mutational strategies of native IDPs with sweeping residue level or domain level mutations.
What is needed are peptide biopolymers comprising intrinsically disordered proteins that exhibit controlled phase separation based on their amino acid sequence, aromatic:aliphatic ratio, hydrophobicity, temperature, molecular weight, and concentration.
One embodiment described herein is a polypeptide with controlled reversible phase separation comprising ten or more repeats of an amino acid sequence comprising: (X-Z1-X-Z2-Z3-X-Z4-Z3)n, where: X is proline (P) or glycine (G) and the ratio of P:G is any number, Z1 is arginine (R), aspartic acid (D), or lysine (K) and the ratio of R:D is any number and the ratio of K:R can be any number; Z2 is Asp (D), Arg (R), Glu (E), where the ratio of R:D can be any number and D:E can be any number; Z3 is asparagine (N), glutamine (Q), serine (S), or threonine (T) were the ratio among N:Q:S:T can be any number, and Z4 is tyrosine (Y), histidine (H), tryptophan (W), phenylalanine (F), methionine (M), valine (V), isoleucine (I), alanine (A), or leucine (L) and the ratio among Y:H:W:F:M:V:I:A:L can be any number. In one aspect, X is proline (P) or glycine (G) and the ratio of P:G is between 1:3 and 3:1. In another aspect, Z1 is arginine (R), aspartic acid (D), or lysine (K) and the ratio of R:D does not exceed 1:5 and the ratio of K:R can be any number. In another aspect, the phase separation is dependent on temperature, molecular weight, hydrophobicity, aromatic:aliphatic ratio, and concentration. In another aspect, n is 10 to 200. In another aspect, molecular weight is at least 5 kDa to 500 kDa. In another aspect, the molecular weight is about 5 kDa to about 100 kDa. In another aspect, the phase separation temperature is 0 to 100° C. In another aspect, the phase separation temperature is 4 to 25° C.; ˜25° C.; 25 to 37° C.; ˜37° C.; 35 to 38° C.; or >38° C. In another aspect, the polypeptide comprises modified amino acids, a reporter protein, or an enzyme. In another aspect, the sequence comprises: (G-R-G-D-S-P-Y-S)m, where m is 20 to 80. In another aspect, the polypeptide comprises a sequence selected from one or more of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, or 197-279, or combinations thereof.
Another embodiment described herein is a pharmaceutically acceptable composition comprising a polypeptide with controlled reversible phase separation comprising ten or more repeats of an amino acid sequence comprising: (X-Z1-X-Z2-Z3-X-Z4-Z3)n, where: X is proline (P) or glycine (G) and the ratio of P:G is any number; Z1 is arginine (R), aspartic acid (D), or lysine (K) and the ratio of R:D is any number and the ratio of K:R can be any number; Z2 is Asp (D), Arg (R), Glu (E), where the ratio of R:D can be any number and D:E can be any number; Z3 is asparagine (N), glutamine (Q), serine (S), or threonine (T) were the ratio among N:Q:S:T can be any number; and Z4 is tyrosine (Y), histidine (H), tryptophan (W), phenylalanine (F), methionine (M), valine (V), isoleucine (I), alanine (A), or leucine (L) and the ratio among Y:H:W:F:M:V:I:A:L can be any number. In one aspect, X is proline (P) or glycine (G) and the ratio of P:G is between 1:3 and 3:1. In another aspect, Z1 is arginine (R), aspartic acid (D), or lysine (K) and the ratio of R:D does not exceed 1:5 and the ratio of K:R can be any number. In another aspect, the composition further comprises an attached molecule comprising one or more of an antibody binding domain derived from Staphylococcus protein A (ZD) (SEQ ID NO:159), an antimicrobial peptide selected from LL37 (SEQ ID NO: 161), Ib-M1 (SEQ ID NO: 163), Ib-M2 (SEQ ID NO: 165), Ib-M5 (SEQ ID NO: 167), Cathelecidin-1 (SEQ ID NO: 169), A(A1R, A8R, I17K) (SEQ ID NO: 171), H5 (SEQ ID NO: 173), H5-61-90 (SEQ ID NO: 175); RGD peptide (RGDSPAS, SEQ ID NO: 39); protein drugs, GLP-1 (SEQ ID NO: 177); fluorescent reporters (sfGFP (SEQ ID NO: 179), mRuby3 (SEQ ID NO: 181); RNA binding proteins (PUM-HD (SEQ ID NO: 183), eIF4E (SEQ ID NO: 185), PABP (SEQ ID NO: 187), Tis11D (SEQ ID NO: 189)); KH domains (Yifan or FMRP (SEQ ID NO: 191)); or AAV binding peptides PKD1 (SEQ ID NO: 193) or PKD2 (SEQ ID NO: 195). In another aspect, the composition enhances bioavailability of the attached molecule as compared to the free form of the attached molecule. In another aspect, the composition enhances expression of the attached molecule as compared to the free form of the attached molecule. In another aspect, the composition enhances the stability of the attached molecule as compared to the free form of the attached molecule. In another aspect, the composition enhances stability of the attached molecule during prokaryotic and eukaryotic expression as compared to the free form of the attached molecule. In another aspect, the enhanced stability includes resistance to denaturation during freezing, thawing, or lyophilization. In another aspect, the composition modulates enzymatic, metabolic, or physiological functions within cells or organisms. In another aspect, the modulation reduces bioavailability of the attached molecules. In another aspect, the attached molecules comprise therapeutic or cytotoxic proteins or peptides.
Another embodiment described herein is a method for enhancing the bioavailability or stability of a protein, the method comprising creating a fusion protein of one or more proteins and a polypeptide with controlled reversible phase separation comprising ten or more repeats of an amino acid sequence comprising: (X-Z1-X-Z2-Z3-X-Z4-Z3)n, where: X is proline (P) or glycine (G) and the ratio of P:G is any number; Z1 is arginine (R), aspartic acid (D), or lysine (K) and the ratio of R:D is any number and the ratio of K:R can be any number; Z2 is Asp (D), Arg (R), Glu (E), where the ratio of R:D can be any number and D:E can be any number Z3 is asparagine (N), glutamine (Q), serine (S), or threonine (T) were the ratio among N:Q:S:T can be any number; and Z4 is tyrosine (Y), histidine (H), tryptophan (W), phenylalanine (F), methionine (M), valine (V), isoleucine (I), alanine (A), or leucine (L) and the ratio among Y:H:W:F:M:V:I:A:L can be any number. In one aspect, X is proline (P) or glycine (G) and the ratio of P:G is between 1:3 and 3:1. In another aspect, Z1 is arginine (R), aspartic acid (D), or lysine (K) and the ratio of R:D does not exceed 1:5 and the ratio of K:R can be any number. In one aspect, X is proline (P) or glycine (G) and the ratio of P:G is between 1:3 and 3:1. In another aspect, Z1 is arginine (R), aspartic acid (D), or lysine (K) and the ratio of R:D does not exceed 1:5 and the ratio of K:R can be any number. In another aspect, the protein comprises one or more of an antibody binding domain derived from Staphylococcus protein A (ZD) (SEQ ID NO:159), an antimicrobial peptide selected from LL37 (SEQ ID NO: 161), Ib-M1 (SEQ ID NO: 163), Ib-M2 (SEQ ID NO: 165), Ib-M5 (SEQ ID NO: 167), Cathelecidin-1 (SEQ ID NO: 169), A(A1R, A8R, I17K) (SEQ ID NO: 171), H5 (SEQ ID NO: 173), H5-61-90 (SEQ ID NO: 175); RGD peptide (RGDSPAS, SEQ ID NO: 39); protein drugs, GLP-1 (SEQ ID NO: 177); fluorescent reporters (sfGFP (SEQ ID NO: 179), mRuby3 (SEQ ID NO: 181); RNA binding proteins (PUM-HD (SEQ ID NO: 183), eIF4E (SEQ ID NO: 185), PABP (SEQ ID NO: 187), Tis11D (SEQ ID NO: 189)); KH domains (Yifan or FMRP (SEQ ID NO: 191)); or AAV binding peptides PKD1 (SEQ ID NO: 193) or PKD2 (SEQ ID NO: 195). In another aspect, the enhanced bioavailability of the fusion protein can be used for isolation or separation of a biologic molecule. In another aspect, the biologic molecule comprises one or more of a lipid, a cell, a protein, a nucleic acid, a carbohydrate, or a viral particle. In another aspect, the nucleic acid is single stranded or double stranded DNA or RNA. In another aspect, the viral particle is an adenovirus particle, an adeno-associated virus particle, a lentivirus particle, a retrovirus particle, a poxvirus particle, a measle virus particle, or herpesvirus particle. In another aspect, the protein comprises albumin, monoclonal IgG antibodies, or Fc fusion antibodies. In another aspect, the isolation or separation is accomplished via reversible phase separation.
This patent or application contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.
The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and,” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.
For the specification of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
The term “about” as used herein as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain aspects, the term “about” refers to a range of values that fall within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
“Affinity” refers to the binding strength of a binding polypeptide to its target (i.e., binding partner).
“Agonist” refers to an entity that binds to a receptor and activates the receptor to produce a biological response. An “antagonist” blocks or inhibits the action or signaling of the agonist. An “inverse agonist” causes an action opposite to that of the agonist. The activities of agonists, antagonists, and inverse agonists may be determined in vitro, in situ, in vivo, or a combination thereof.
“Amino acid” as used herein refers to naturally occurring and non-natural synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code. Amino acids can be referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Amino acids include the side chain and polypeptide backbone portions.
As used herein, the term “biomarker” refers to a naturally occurring biological molecule present in a subject at varying concentrations that is useful in identifying and/or classifying a disease or a condition. The biomarker can include genes, proteins, polynucleotides, nucleic acids, ribonucleic acids, polypeptides, or other biological molecules used as an indicator or marker for disease. In some embodiments, the biomarker comprises a disease marker. For example, the biomarker can be a gene that is upregulated or downregulated in a subject that has a disease. As another example, the biomarker can be a polypeptide whose level is increased or decreased in a subject that has a disease or risk of developing a disease. In some embodiments, the biomarker comprises a small molecule. In some embodiments, the biomarker comprises a polypeptide.
The terms “control,” “reference level,” and “reference” are used herein interchangeably. The reference level may be a predetermined value or range, which is employed as a benchmark against which to assess the measured result. “Control group” as used herein refers to a group of control subjects. The predetermined level may be a cutoff value from a control group. The predetermined level may be an average from a control group. Cutoff values (or predetermined cutoff values) may be determined by Adaptive Index Model (AIM) methodology. Cutoff values (or predetermined cutoff values) may be determined by a receiver operating curve (ROC) analysis from biological samples of the patient group. ROC analysis, as generally known in the biological arts, is a determination of the ability of a test to discriminate one condition from another, e.g., to determine the performance of each marker in identifying a patient having CRC. A description of ROC analysis is provided in P. J. Heagerty et al. (Biometrics 2000, 56, 337-44), the disclosure of which is hereby incorporated by reference in its entirety. Alternatively, cutoff values may be determined by a quartile analysis of biological samples of a patient group. For example, a cutoff value may be determined by selecting a value that corresponds to any value in the 25th-75th percentile range, preferably a value that corresponds to the 25th percentile, the 50th percentile or the 75th percentile, and more preferably the 75th percentile. Such statistical analyses may be performed using any method known in the art and can be implemented through any number of commercially available software packages (e.g., from Analyse-it Software Ltd., Leeds, UK; StataCorp LP, College Station, Tex.; SAS Institute Inc., Cary, N.C.). The healthy or normal levels or ranges for a target or for a protein activity may be defined in accordance with standard practice.
The term “expression vector” indicates a plasmid, a virus, or another medium, known in the art, into which a nucleic acid sequence for encoding a desired protein can be inserted or introduced.
The term “host cell” is a cell that is susceptible to transformation, transfection, transduction, conjugation, and the like with a nucleic acid construct or expression vector. Host cells can be derived from plants, bacteria, yeast, fungi, insects, animals, etc. In some embodiments, the host cell includes Escherichia coli.
“Polymer” as used herein is intended to encompass a homopolymer, heteropolymer, block polymer, co-polymer, ter-polymer, etc., and blends, combinations, and mixtures thereof. Examples of polymers include, but are not limited to, functionalized polymers, such as a polymer comprising 5-vinyltetrazole monomer units and having a molecular weight distribution less than 2.0. The polymer may be or contain one or more of a star block copolymer, a linear polymer, a branched polymer, a hyperbranched polymer, a dendritic polymer, a comb polymer, a graft polymer, a brush polymer, a bottle-brush copolymer and a crosslinked structure, such as a block copolymer comprising a block of 5-vinyltetrazole monomer units. Polymers include, without limitation, polyesters, poly(meth)acrylamides, poly(meth)acrylates, polyethers, polystyrenes, polynorbonenes and monomers that have unsaturated bonds. For example, amphiphilic comb polymers are described in U.S. Patent Application Publication No. US 2007/0087114 and in U.S. Pat. No. 6,207,749 to Mayes et al., the disclosure of each of which is herein incorporated by reference in its entirety. The amphiphilic comb-type polymers may be present in the form of copolymers, containing a backbone formed of a hydrophobic, water-insoluble polymer and side chains formed of short, hydrophilic non-cell binding polymers. Examples of other polymers include, but are not limited to, polyalkylenes such as polyethylene and polypropylene; polychloroprene; polyvinyl ethers; such as polyvinyl acetate); polyvinyl halides such as polyvinyl chloride); polysiloxanes; polystyrenes; polyurethanes; polyacrylates; such as poly(methyl (meth)acrylate), poly(ethyl (meth)acrylate), poly(n-butyl(meth)acrylate), poly(isobutyl (meth)acrylate), poly(tert-butyl (meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl (meth)acrylate), poly(lauryl (meth)acrylate), poly(phenyl (meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate); polyacrylamides such as poly(acrylamide), poly(methacrylamide), poly(ethyl acrylamide), poly(ethyl methacrylamide), poly(N-isopropyl acrylamide), poly(n, iso, and tert-butyl acrylamide); and copolymers and mixtures thereof. These polymers may include useful derivatives, including polymers having substitutions, additions of chemical groups, for example, alkyl groups, alkylene groups, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art. The polymers may include zwitterionic polymers such as, for example, polyphosphorycholine, polycarboxybetaine, and polysulfobetaine. The polymers may have side chains of betaine, carboxybetaine, sulfobetaine, oligoethylene glycol (OEG), sarcosine, or polyethyleneglycol (PEG). For example, poly(oligoethyleneglycol methacrylate) (poly(OEGMA)) may be used. Poly(OEGMA) may be hydrophilic, water-soluble, non-fouling, non-toxic and non-immunogenic due to the OEG side chains.
“Polynucleotide” as used herein can be single stranded or double stranded or can contain portions of both double stranded and single stranded sequence. The polynucleotide can be nucleic acid, natural or synthetic, DNA, genomic DNA, cDNA, RNA, or a hybrid, where the polynucleotide can contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, and isoguanine. Polynucleotides can be obtained by chemical synthesis methods or by recombinant methods.
A “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds. The polypeptide can be natural, synthetic, or a modification or combination of natural and synthetic. Peptides and polypeptides include proteins such as binding proteins, receptors, and antibodies. The terms “polypeptide,” “protein,” and “peptide” are used interchangeably herein. “Primary structure” refers to the amino acid sequence of a particular peptide. “Secondary structure” refers to locally ordered, three-dimensional structures within a polypeptide. These structures are commonly known as domains, e.g., enzymatic domains, extracellular domains, transmembrane domains, pore domains, and cytoplasmic tail domains. Domains are portions of a polypeptide that form a compact unit of the polypeptide and are typically 15 to 350 amino acids long. Exemplary domains include domains with enzymatic activity or ligand binding activity. Typical domains are made up of sections of lesser organization such as stretches of beta-sheet and alpha-helices. “Tertiary structure” refers to the complete three-dimensional structure of a polypeptide monomer. “Quaternary structure” refers to the three-dimensional structure formed by the noncovalent association of independent tertiary units.
“Reporter,” “reporter group,” “label,” and “detectable label” are used interchangeably herein. The reporter is capable of generating a detectable signal. The label can produce a signal that is detectable by visual or instrumental means. A variety of reporter groups can be used, differing in the physical nature of signal transduction (e.g., fluorescence, electrochemical, nuclear magnetic resonance (NMR), and electron paramagnetic resonance (EPR)) and in the chemical nature of the reporter group. Various reporters include signal-producing substances, such as chromagens, fluorescent compounds, chemiluminescent compounds, radioactive compounds, and the like. In some embodiments, the reporter comprises a radiolabel. Reporters may include moieties that produce light, e.g., acridinium compounds, and moieties that produce fluorescence, e.g., fluorescein. In some embodiments, the signal from the reporter is a fluorescent signal. The reporter may comprise a fluorophore. Examples of fluorophores include, but are not limited to, acrylodan (6-acryloyl-2-dimethylaminonaphthalene), badan (6-bromo-acetyl-2-dimethylamino-naphthalene), rhodamine, naphthalene, danzyl aziridine, 4-[N-[(2-iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole ester (IANBDE), 4-[N-[(2-iodoacetoxy)ethyl]-N-methylamino-7-nitrobenz-2-oxa-1,3-diazole (IANBDA), fluorescein, dipyrrometheneboron difluoride (BODIPY), 4-nitrobenzo[c][1,2,5]oxadiazole (NBD), Alexa fluorescent dyes, and derivatives thereof. Fluorescein derivatives may include, for example, 5-fluorescein, 6-carboxyfluorescein, 3′6-carboxyfluorescein, 5(6)-carboxyfluorescein, 6-hexachlorofluorescein, 6-tetrachlorofluorescein, fluorescein, and isothiocyanate.
“Sample” or “test sample” as used herein can mean any sample in which the presence and/or level of a target is to be detected or determined. Samples may include liquids, solutions, emulsions, or suspensions. Samples may include a medical sample. Samples may include any biological fluid or tissue, such as blood, whole blood, fractions of blood such as plasma and serum, muscle, interstitial fluid, sweat, saliva, urine, tears, synovial fluid, bone marrow, cerebrospinal fluid, nasal secretions, sputum, amniotic fluid, bronchoalveolar lavage fluid, gastric lavage, emesis, fecal matter, lung tissue, peripheral blood mononuclear cells, total white blood cells, lymph node cells, spleen cells, tonsil cells, cancer cells, tumor cells, bile, digestive fluid, skin, or combinations thereof. In some embodiments, the sample comprises an aliquot. In other embodiments, the sample comprises a biological fluid. Samples can be obtained by any means known in the art. The sample can be used directly as obtained from a patient or can be pre-treated, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, and the like, to modify the character of the sample in some manner as discussed herein or otherwise as is known in the art.
The term “sensitivity” as used herein refers to the number of true positives divided by the number of true positives plus the number of false negatives, where sensitivity (“sens”) may be within the range of 0<sens<1. Ideally, method embodiments herein have the number of false negatives equaling zero or close to equaling zero, so that no subject is wrongly identified as not having a disease when they indeed have the disease. Conversely, an assessment often is made of the ability of a prediction algorithm to classify negatives correctly, a complementary measurement to sensitivity.
The term “specificity” as used herein refers to the number of true negatives divided by the number of true negatives plus the number of false positives, where specificity (“spec”) may be within the range of 0<spec<1. Ideally, the methods described herein have the number of false positives equaling zero or close to equaling zero, so that no subject is wrongly identified as having a disease when they do not in fact have disease. Hence, a method that has both sensitivity and specificity equaling one, or 100%, is preferred.
By “specifically binds,” it is generally meant that a polypeptide binds to a target when it binds to that target more readily than it would bind to a random, unrelated target.
“Subject” as used herein can mean a mammal that wants or is in need of the herein described peptide biopolymers comprising one or more fusion proteins. The subject may be a human or a non-human animal. The subject may be a mammal. The mammal may be a primate or a non-primate. The mammal can be a primate such as a human; a non-primate such as, for example, dog, cat, horse, cow, pig, mouse, rat, camel, llama, goat, rabbit, sheep, hamster, and guinea pig; or non-human primate such as, for example, monkey, chimpanzee, gorilla, orangutan, and gibbon. The subject may be of any age or stage of development, such as, for example, an adult, an adolescent, or an infant.
“Transition” or “phase transition” refers to the aggregation of the thermally responsive polypeptides. Phase transition occurs sharply and reversibly at a specific temperature called the lower critical solution temperature (LCST) or the inverse transition temperature TA. Below the transition temperature, the thermally responsive polypeptide (or a polypeptide comprising a thermally responsive polypeptide) is highly soluble. Upon heating past the transition temperature, the thermally responsive polypeptides hydrophobically collapse and aggregate, forming a separate, gel-like phase. “Inverse transition cycling” refers to a protein purification method for thermally responsive polypeptides (or a polypeptide comprising a thermally responsive polypeptide). The protein purification method may involve the use of thermally responsive polypeptide's reversible phase transition behavior to cycle the solution through soluble and insoluble phases, thereby removing contaminants.
“Treatment” or “treating,” when referring to protection of a subject from a disease, means preventing, suppressing, repressing, ameliorating, or eliminating the disease. Preventing the disease involves administering a composition of the present invention to a subject prior to onset of the disease. Suppressing the disease involves administering a composition of the present invention to a subject after induction of the disease but before its clinical appearance. Repressing or ameliorating the disease involves administering a composition of the present invention to a subject after clinical appearance of the disease.
“Substantially identical” can mean that a first and second amino acid sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% over a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 or greater number of amino acids.
“Valency” as used herein refers to the potential binding units or binding sites. The term “multivalent” refers to multiple potential binding units. The terms “multimeric” and “multivalent” are used interchangeably herein.
“Variant” used herein with respect to a polynucleotide means (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a polynucleotide that is substantially identical to a referenced polynucleotide or the complement thereof; or (iv) a polynucleotide that hybridizes under stringent conditions to the referenced polynucleotide, complement thereof, or a sequences substantially identical thereto.
A “variant” can further be defined as a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Representative examples of “biological activity” include the ability to be bound by a specific antibody or polypeptide or to promote an immune response. Variant can mean a substantially identical sequence. Variant can mean a functional fragment thereof. Variant can also mean multiple copies of a polypeptide. The multiple copies can be in tandem or separated by a linker. Variant can also mean a polypeptide with an amino acid sequence that is substantially identical to a referenced polypeptide with an amino acid sequence that retains at least one biological activity. A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree, and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids. See Kyte et al., J. Mol. Biol. 1982, 757, 105-132. The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and retain protein function. In one aspect, amino acids having hydropathic indices of ±2 are substituted. The hydrophobicity of amino acids can also be used to reveal substitutions that would result in polypeptides retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a polypeptide permits calculation of the greatest local average hydrophilicity of that polypeptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity, as discussed in U.S. Pat. No. 4,554,101, which is incorporated herein by reference. Substitution of amino acids having similar hydrophilicity values can result in polypeptides retaining biological activity, for example immunogenicity, as is understood in the art. Substitutions can be performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.
A variant can be a polynucleotide sequence that is substantially identical over the full length of the full gene sequence or a fragment thereof. The polynucleotide sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the gene sequence or a fragment thereof. A variant can be an amino acid sequence that is substantially identical over the full length of the amino acid sequence or fragment thereof. The amino acid sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the amino acid sequence or a fragment thereof.
The term “fusion protein” as described herein at least one intrinsically disordered polypeptide and at least one other polypeptide. The fusion protein may optionally include at least one linker. In one aspect, the intrinsically disordered polypeptide has controlled reversible phase separation.
In some embodiments, the fusion protein includes more than one polypeptide with controlled reversible phase separation. The polypeptide with controlled reversible phase separation can include multiple repeats of a peptide motif. The fusion protein may include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, and least 40, at least 60, at least 80, at least 120, at least 160, or at least 200 polypeptides with controlled reversible phase separation or repeats of a peptide motif with controlled reversible phase separation. The fusion protein may include less than 30, less than 25, or less than 20 polypeptides with controlled reversible phase separation or repeats of a peptide motif. The fusion protein may include between 1 and 160, between 1 and 80, between 1 and 60, between 1 and 40, between 1 and 20, or between 1 and 10 polypeptides with controlled reversible phase separation or repeats of a peptide motif. In such embodiments, the polypeptides with controlled reversible phase separation may be the same or different from one another. In some embodiments, the fusion protein includes more than one polypeptide with controlled reversible phase separation positioned in tandem to one another (e.g., repeats of a peptide motif).
In some embodiments, the fusion protein includes one or more binding polypeptide. The fusion protein may include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 binding polypeptides. The fusion protein may include less than 30, less than 25, less than 20, less than 10, or less than 5 binding polypeptides. The fusion protein may include between 1 and 30, between 1 and 20, or between 1 and 10 binding polypeptides. In such embodiments, the binding polypeptides may be the same or different from one another. In some embodiments, the fusion protein includes more than one binding polypeptide positioned in tandem to one another. In some embodiments, the fusion protein includes 2 to 6 binding polypeptides. In some embodiments, the fusion protein includes two binding polypeptides. In some embodiments, the fusion protein includes three binding polypeptides. In some embodiments, the fusion protein includes four binding polypeptides. In some embodiments, the fusion protein includes five binding polypeptides. In some embodiments, the fusion protein includes six binding polypeptides.
The fusion protein may be expressed recombinantly in a host cell according to one of ordinary skill in the art. The fusion protein may be purified by any means known to one of skill in the art. For example, the fusion protein may be purified using chromatography, such as liquid chromatography, size exclusion chromatography, or affinity chromatography, or a combination thereof. In some embodiments, the fusion protein is purified without chromatography. In some embodiments, the fusion protein is purified using inverse transition cycling.
Polypeptides with Controlled Reversible Phase Separation
The polypeptides with controlled reversible phase separation may comprise any polypeptide that has minimal or no secondary structure as observed by CD, being soluble at a temperature below its lower critical solution temperature (LCST) and/or at a temperature above its upper critical solution temperature (UCST), and comprising a repeated amino acid sequence. LCST is the temperature below which the polypeptide is miscible. UCST is the temperature above which the polypeptide is miscible. In some embodiments, the polypeptide with controlled reversible phase separation has only UCST behavior. In some embodiments, the polypeptide with controlled reversible phase separation has only LCST behavior. In some embodiments, the polypeptide with controlled reversible phase separation has both UCST and LCST behavior. The polypeptide with controlled reversible phase separation may comprise a repeated sequence of amino acids. The polypeptides with controlled reversible phase separation may have a LCST between about 0° C. and about 100° C., between about 10° C. and about 50° C., or between about 20° C. and about 42° C. The polypeptide with controlled reversible phase separation may have a UCST between about 0° C. and about 100° C., between about 10° C. and about 50° C., or between about 20° C. and about 42° C. In some embodiments, the polypeptide with controlled reversible phase separation has a transition temperature between room temperature (about 25° C.) and body temperature (about 37° C.). In some embodiments, a fusion protein comprising one or more thermally responsive polypeptides has a transition temperature between room temperature (about 25° C.) and body temperature (about 37° C.). In some embodiments, the polypeptide with controlled reversible phase separation has no LCST or UCST behavior. The polypeptide with controlled reversible phase separation may have its LCST or UCST below body temperature or above body temperature at the concentration at which the peptide biopolymer comprising one or more fusion proteins is administered to a subject.
In some embodiments, the polypeptide with controlled reversible phase separation comprises one or more thermally responsive polypeptides. Thermally responsive polypeptides may include, for example, elastin-like polypeptides (ELP) and resilin-like protein (RLP).
In some embodiment, the polypeptide with controlled reversible phase separation comprises a plurality of polypeptides with controlled reversible phase separation. In one aspect, the polypeptide with controlled reversible phase separation a di-block of two or more polypeptides with controlled reversible phase separation. In one aspect, the polypeptides with controlled reversible phase separation comprise a di-block of a resilin-like protein (RLP) and an elastin-like polypeptide (ELP).
In one embodiment, the polypeptide with controlled reversible phase separation comprises one or more core polypeptides. In one aspect, the core polypeptide is a resilin-like polypeptide (RLP). RLPs are derived from arthropod Rec1-resilin. Rec1-resilin is environmentally responsive and exhibits a dual phase transition behavior. The thermally responsive RLPs can have LCST and UCST. Additional examples of suitable thermally responsive polypeptides are described in U.S. Patent Application Publication Nos. US 2012/0121709, and US 2015/0112022, each of which is incorporated herein by reference. In one embodiment, the RLP polypeptide comprises the sequence (GRGDSPYS)n (SEQ ID NO: 1). The polypeptide with controlled reversible phase separation may comprise an amino acid sequence comprising (G1-R2-G3-D4-S5-P6-Y7-S8))n, where n is 20-200. In some embodiments, n is 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300. In some embodiments, n may be less than 500, less than 400, less than 300, less than 200, or less than 100. In some embodiments, n may be between 1 and 500, between 1 and 400, between 1 and 300, or between 1 and 200. In some embodiments, n is 20, 40, 60, 80, 100, 120, 160, 180, or 200. In one aspect, n is 20 to 200 repeats. In one aspect, n is 20 to 60 repeats.
Thermally responsive polypeptides may have a phase transition. The thermally responsive polypeptide may impart a phase transition characteristic to an unstructured polypeptide or fusion protein. “Phase transition” or “transition” may refer to the aggregation of the thermally responsive polypeptide, which occurs sharply and reversibly at a specific temperature called the lower critical solution temperature (LCST) or the inverse transition temperature (T1). Below the transition temperature (LCST or T1), the thermally responsive polypeptides, (or polypeptides comprising a thermally responsive polypeptide) may be highly soluble. Upon heating above the transition temperature, thermally responsive polypeptides hydrophobically may collapse and aggregate, forming a separate, gel-like phase.
The thermally responsive polypeptides can phase transition at a variety of temperatures and concentrations. Thermally responsive polypeptides, for example, may not affect the binding or potency of the binding polypeptides. Thermally responsive polypeptides may allow the fusion protein to be tuned by a user to any number of desired transition temperatures, molecular weights, and formats.
Thermally responsive polypeptides may exhibit inverse phase transition behavior and thus, the fusion protein comprising the thermally responsive polypeptide may exhibit inverse phase transition behavior. Inverse phase transition behavior may be used to form drug depots within a tissue of a subject for controlled (slow) release of the fusion protein. Inverse phase transition behavior may also enable purification of the fusion protein using inverse transition cycling, thereby eliminating the need for chromatography.
One embodiment described herein is a polypeptide with controlled reversible phase separation comprising ten or more repeats of an amino acid sequence comprising:
(X-Z1-X-Z2-Z3-X-Z4-Z3)n,
where:
The binding polypeptide (or “targeting polypeptide”) may comprise any polypeptide that is capable of binding at least one target. The binding polypeptide may bind at least one target. “Target” may be an entity capable of being bound by the binding polypeptide. Targets may include, for example, another polypeptide, a cell surface receptor, a carbohydrate, an antibody, a small molecule, or a combination thereof. The target may be a biomarker. The target may be activated through agonism or blocked through antagonism. The binding polypeptide may specifically bind the target. By binding target, the binding polypeptide may act as a targeting moiety, an agonist, an antagonist, or a combination thereof. In some embodiments, the binding polypeptide domain binds
The binding polypeptide may be a monomer that binds to a target. The monomer may bind one or more targets. The binding polypeptide may form an oligomer. The binding polypeptide may form an oligomer with the same or different binding polypeptides. The oligomer may bind to a target. The oligomer may bind one or more targets. One or more monomers within an oligomer may bind one or more targets. In some embodiments, the fusion protein is multivalent. In some embodiments, the fusion protein binds multiple targets. In some embodiments, the activity of the binding polypeptide alone is the same as the activity of the binding protein when part of a fusion protein.
In one aspect, the wherein the binding polypeptide comprises one or more of an antibody binding domain derived from Staphylococcus protein A (ZD) (SEQ ID NO:159), an antimicrobial peptide selected from LL37 (SEQ ID NO: 161), Ib-M1 (SEQ ID NO: 163), Ib-M2 (SEQ ID NO: 165), Ib-M5 (SEQ ID NO: 167), Cathelecidin-1 (SEQ ID NO: 169), A(A1R, A8R, I17K) (SEQ ID NO: 171), H5 (SEQ ID NO: 173), H5-61-90 (SEQ ID NO: 175); RGD peptide (RGDSPAS, SEQ ID NO: 39); protein drugs, GLP-1 (SEQ ID NO: 177); fluorescent reporters (sfGFP (SEQ ID NO: 179), mRuby3 (SEQ ID NO: 181); RNA binding proteins (PUM-HD (SEQ ID NO: 183), eIF4E (SEQ ID NO: 185), PABP (SEQ ID NO: 187), Tis11D (SEQ ID NO: 189)); KH domains (Yifan or FMRP (SEQ ID NO: 191)); or AAV binding peptides PKD1 (SEQ ID NO: 193) or PKD2 (SEQ ID NO: 195).
In some embodiments, the fusion protein further includes at least one linker. In some embodiments, the fusion protein includes more than one linker. In such embodiments, the linkers may be the same or different from one another. The fusion protein may include, none, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 linkers. The fusion protein may include less than 500, less than 400, less than 300, or less than 200 linkers. The fusion protein may include between 1 and 1000, between 10 and 900, between 10 and 800, or between 5 and 500 linkers.
The linker may be positioned in between a binding polypeptide and a polypeptide with controlled reversible phase separation, in between binding polypeptides, in between polypeptides with controlled reversible phase separation, or a combination thereof. Multiple linkers may be positioned adjacent to one another. Multiple linkers may be positioned adjacent to one another and in between the binding polypeptide and the polypeptide with controlled reversible phase separation.
The linker may be a polypeptide of any amino acid sequence and length. The linker may act as a spacer peptide. The linker may occur between polypeptide domains. The linker may sufficiently separate the binding domains of the binding polypeptide while preserving the activity of the binding domains. In some embodiments, the linker comprises charged amino acids. In some embodiments, the linker is flexible. In some embodiments, the linker comprises at least one glycine and at least one serine. In some embodiments, the linker comprises at least one proline.
Further provided are polynucleotides encoding the fusion proteins detailed herein. A vector may include the polynucleotide encoding the fusion proteins detailed herein. To obtain expression of a polypeptide, one typically subclones the polynucleotide encoding the polypeptide into an expression vector that contains a promoter to direct transcription, a transcription/translation terminator, and if for a nucleic acid encoding a protein, a ribosome binding site for translational initiation. An example of a vector is pET24. Suitable bacterial promoters are well known in the art. Further provided is a host cell transformed or transfected with an expression vector comprising a polynucleotide encoding a fusion protein as detailed herein. Bacterial expression systems for expressing the protein are available in, e.g., E. coli, Bacillus sp., and Salmonella (Paiva et al., Gene 1983, 22, 229-235; Mosbach et al., Nature 1983, 302, 543-545). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are commercially available. Retroviral expression systems can be used in the present invention. In some embodiments, the fusion protein comprises repeats or single sequences of one or more of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, or 197-279. In some embodiments, the fusion protein comprises repeats or single sequences of one or more of a polypeptide encoded by a polynucleotide sequence of any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, or 158. In some embodiments, the fusion protein comprises a polypeptide comprising an amino acid sequence of any one of SEQ ID NO: 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, or 316.
The peptide biopolymers comprising one or more fusion proteins as detailed herein can be formulated in accordance with standard techniques well known to those skilled in the pharmaceutical art to form a therapeutic agent or targeted delivery agent. Such compositions comprising peptide biopolymers comprising one or more fusion proteins can be administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular subject, and the route of administration.
The peptide biopolymers comprising one or more fusion proteins can be administered prophylactically or therapeutically. In prophylactic administration, the peptide biopolymer can be administered in an amount sufficient to induce a response. In therapeutic applications, the peptide biopolymers are administered to a subject in need thereof in an amount sufficient to elicit a therapeutic effect. An amount adequate to accomplish this is defined as “therapeutically effective dose.” Amounts effective for this use will depend on, e.g., the particular composition of the peptide biopolymer regimen administered, the manner of administration, the stage, and severity of the disease, the general state of health of the patient, and the judgment of the prescribing physician.
The peptide biopolymer can be administered by methods well known in the art as described in Donnelly et al. Ann. Rev. Immunol. 1997, 75, 617-648; Feigner et al., U.S. Pat. No. 5,580,859; Feigner, U.S. Pat. No. 5,703,055; and Carson et al., U.S. Pat. No. 5,679,647, the contents of each of which are incorporated herein by reference in their entirety. The peptide biopolymer can be complexed to particles or beads that can be administered to an individual, for example, using a vaccine gun. One skilled in the art would know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration.
The peptide biopolymers can be delivered via a variety of routes. Typical delivery routes include parenteral administration, e.g., intradermal, intramuscular, or subcutaneous delivery. Other routes include oral administration, intranasal, intravaginal, transdermal, intravenous, intraarterial, intratumoral, intraperitoneal, and epidermal routes. In some embodiments, the peptide biopolymer is administered intravenously, intraarterially, or intraperitoneally to the subject.
The peptide biopolymer can be a liquid preparation such as a suspension, syrup, or elixir. The peptide biopolymer can be incorporated into liposomes, microspheres, or other polymer matrices (such as by a method described in Feigner et al., U.S. Pat. No. 5,703,055; Gregoriadis, Liposome Technology, Vols. I to III (2nd ed. 1993), the contents of which are incorporated herein by reference in their entirety). Liposomes can consist of phospholipids or other lipids, and can be nontoxic, physiologically acceptable, and metabolizable carriers that are relatively simple to make and administer.
In some embodiments, the peptide biopolymer is administered in a controlled release formulation. In some embodiments, the peptide biopolymer comprises one or more thermally responsive polypeptides, the thermally responsive polypeptide having a transition temperature such that the peptide biopolymer remains soluble prior to administration and such that the peptide biopolymer transitions upon administration to a gel-like depot in the subject. In some embodiments, the peptide biopolymer comprises one or more fusion proteins comprising one or more thermally responsive polypeptides, the thermally responsive polypeptide having a transition temperature such that the fusion protein remains soluble at room temperature and such that the fusion protein transitions upon administration to a gel-like depot in the subject. For example, in some embodiments, the fusion protein comprises one or more thermally responsive polypeptides, the thermally responsive polypeptide having a transition temperature between room temperature (about 25° C.) and body temperature (about 37° C.), whereby the fusion protein can be administered to form a depot. As used herein, “depot” refers to a gel-like composition comprising a fusion protein that releases the fusion protein over time. In some embodiments, the peptide biopolymer can be injected subcutaneously or intratumorally to form a depot (coacervate). The depot may provide controlled (slow) release of the peptide biopolymer. The depot may provide slow release of the peptide biopolymer into the circulation or the tumor, for example. In some embodiments, the peptide biopolymer may be released from the depot over a period of at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 1 week, at least about 1.5 weeks, at least about 2 weeks, at least about 2.5 weeks, at least about 3.5 weeks, at least about 4 weeks, or at least about 1 month.
As used herein, the term “detect” or “determine the presence of” refers to the qualitative measurement of undetectable, low, normal, or high concentrations of one or more peptide biopolymers, targets, or peptide biopolymers bound to target. Detection may include in vitro, ex vivo, or in vivo detection. Detection may include detecting the presence of one or more peptide biopolymers comprising one or more peptide biopolymers or targets versus the absence of the one or more peptide biopolymer or targets. Detection may also include quantification of the level of one or more peptide biopolymers or targets. The terms “quantify,” or “quantification” may be used interchangeably, and may refer to a process of determining the quantity or abundance of a substance (e.g., peptide biopolymer or target), whether relative or absolute. Any suitable method of detection falls within the general scope of the present disclosure. In some embodiments, the peptide biopolymer comprises a reporter attached thereto for detection. In some embodiments, the peptide biopolymer is labeled with a reporter. In some embodiments, detection of a peptide biopolymer bound to a target may be determined by methods including but not limited to, band intensity on a Western blot, flow cytometry, radiolabel imaging, cell binding assays, activity assays, SPR, immunoassay, or by various other methods known in the art.
In some embodiments, including those wherein the peptide biopolymer is an antibody mimic for binding and/or detecting a target, any immunoassay may be utilized. The immunoassay may be an enzyme-linked immunoassay (ELISA), radioimmunoassay (RIA), a competitive inhibition assay, such as forward or reverse competitive inhibition assays, a fluorescence polarization assay, or a competitive binding assay, for example. The ELISA may be a sandwich ELISA. Specific immunological binding of the f peptide biopolymer to the target can be detected via direct labels, attached to the peptide biopolymer or via indirect labels, such as alkaline phosphatase or horseradish peroxidase. The use of an immobilized peptide biopolymer may be incorporated into the immunoassay. The peptide biopolymers may be immobilized onto a variety of supports, such as magnetic or chromatographic matrix particles, the surface of an assay plate (such as microtiter wells), pieces of a solid substrate material, and the like. An assay strip can be prepared by coating the peptide biopolymer or plurality of peptide biopolymers in an array on a solid support. This strip can then be dipped into the test biological sample and then processed quickly through washes and detection steps to generate a measurable signal, such as a colored spot.
The present invention is directed to a method of treating a disease in a subject in need thereof. The method may comprise administering to the subject an effective amount of the peptide biopolymer comprising one or more peptide biopolymers as described herein. The disease may be selected from cancer, metabolic disease, autoimmune disease, cardiovascular disease, and orthopedic disorders. In some embodiments, the disease is a disease associated with a target of the at least one binding polypeptide.
Metabolic disease may occur when abnormal chemical reactions in the body alter the normal metabolic process. Metabolic diseases may include, for example, insulin resistance, non-alcoholic fatty liver diseases, type 2 diabetes, insulin resistance diseases, cardiovascular diseases, arteriosclerosis, lipid-related metabolic disorders, hyperglycemia, hyperinsulinemia, hyperlipidemia, and glucose metabolic disorders.
Autoimmune diseases arise from an abnormal immune response of the body against substances and tissues normally present in the body. Autoimmune diseases may include, but are not limited to, lupus, rheumatoid arthritis, multiple sclerosis, insulin dependent diabetes mellitis, myasthenia gravis, Grave's disease, autoimmune hemolytic anemia, autoimmune thrombocytopenia purpura, Goodpasture's syndrome, pemphigus vulgaris, acute rheumatic fever, post-streptococcal glomerulonephritis, polyarteritis nodosa, myocarditis, psoriasis, Celiac disease, Crohn's disease, ulcerative colitis, and fibromyalgia.
Cardiovascular disease is a class of diseases that involve the heart or blood vessels. Cardiovascular diseases may include, for example, coronary artery diseases (CAD) such as angina and myocardial infarction (heart attack), stroke, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, heart arrhythmia, congenital heart disease, valvular heart disease, carditis, aortic aneurysms, peripheral artery disease, and venous thrombosis.
Orthopedic disorders or musculoskeletal disorders are injuries or pain in the body's joints, ligaments, muscles, nerves, tendons, and structures that support limbs, neck, and back. Orthopedic disorders may include degenerative diseases and inflammatory conditions that cause pain and impair normal activities. Orthopedic disorders may include, for example, carpal tunnel syndrome, epicondylitis, and tendinitis. Cancers may include, but are not limited to, breast cancer, colorectal cancer, colon cancer, lung cancer, prostate cancer, testicular cancer, brain cancer, skin cancer, rectal cancer, gastric cancer, esophageal cancer, sarcomas, tracheal cancer, head and neck cancer, pancreatic cancer, liver cancer, ovarian cancer, lymphoid cancer, cervical cancer, vulvar cancer, melanoma, mesothelioma, renal cancer, bladder cancer, thyroid cancer, bone cancers, carcinomas, sarcomas, and soft tissue cancers. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is colorectal adenocarcinoma.
One application of protein therapeutics is cancer treatment. In specific embodiments, the present invention provides a method for using scaffold proteins in developing antibody mimetics for oncological targets of interest. With the emergence of scaffold protein engineering come the possibilities for designing potent protein drugs that are unhindered by steric and architectural limitations. Although potent protein drugs can be invaluable for diagnostics or treatments, successful delivery to the target region can pose a great challenge.
Provided herein are methods of diagnosing a disease. The methods may include administering to the subject a peptide biopolymer comprising one or more fusion proteins as described herein and detecting binding of the peptide biopolymer to a target to determine presence of the target in the subject. The presence of the target may indicate the disease in the subject. In other embodiments, the methods may include contacting a sample from the subject with a peptide biopolymer as described herein, determining the level of a target in the sample, and comparing the level of the target in the sample to a control level of the target, wherein a level of the target different from the control level indicates disease in the subject. In some embodiments, the disease is selected from cancer, metabolic disease, autoimmune disease, cardiovascular disease, and orthopedic disorders, as detailed above. In some embodiments, the target comprises a disease marker or biomarker. In some embodiments, the fusion protein may act as an antibody mimic for binding or detecting a target.
Provided herein are methods of determining the presence of a target in a sample. The methods may include contacting the sample with a peptide biopolymer comprising one or more fusion proteins as described herein under conditions to allow a complex to form between the peptide biopolymer and the target in the sample and detecting the presence of the complex. Presence of the complex may be indicative of the target in the sample. In some embodiments, the peptide biopolymer is labeled with a reporter for detection.
In some embodiments, the sample is obtained from a subject and the method further includes diagnosing, prognosticating, or assessing the efficacy of a treatment of the subject. When the method includes assessing the efficacy of a treatment of the subject, then the method may further include modifying the treatment of the subject as needed to improve efficacy.
Provided herein are methods of determining the effectiveness of a treatment for a disease in a subject in need thereof. The methods may include contacting a sample from the subject with a peptide biopolymer comprising a fusion protein as detailed herein under conditions to allow a complex to form between the peptide biopolymer and a target in the sample, determining the level of the complex in the sample, wherein the level of the complex is indicative of the level of the target in the sample, and comparing the level of the target in the sample to a control level of the target, wherein if the level of the target is different from the control level, then the treatment is determined to be effective or ineffective in treating the disease.
Time points may include prior to onset of disease, prior to administration of a therapy, various time points during administration of a therapy, and after a therapy has concluded, or a combination thereof. Upon administration of the peptide biopolymer comprising one or more fusion proteins to the subject, the peptide biopolymer may bind a target, wherein the presence of the target indicates the presence of the disease in the subject at the various time points. In some embodiments, the target comprises a disease marker or biomarker. In some embodiments, the peptide biopolymer may act as an antibody mimic for binding and/or detecting a target. Comparison of the binding of the peptide biopolymer to the target at various time points may indicate whether the disease has progressed, whether the diseased has advanced, whether a therapy is working to treat or prevent the disease, or a combination thereof.
In some embodiments, the control level corresponds to the level in the subject at a time point before or during the period when the subject has begun treatment, and the sample is taken from the subject at a later time point. In some embodiments, the sample is taken from the subject at a time point during the period when the subject is undergoing treatment, and the control level corresponds to a disease-free level or to the level at a time point before the period when the subject has begun treatment. In some embodiments, the method further includes modifying the treatment or administering a different treatment to the subject when the treatment is determined to be ineffective in treating the disease.
It will be apparent to one of ordinary skill in the relevant art that suitable modifications and adaptations to the compositions, formulations, methods, processes, and applications described herein can be made without departing from the scope of any embodiments or aspects thereof. The compositions and methods provided are exemplary and are not intended to limit the scope of any of the specified embodiments. All of the various embodiments, aspects, and options disclosed herein can be combined in any variations or iterations. The scope of the compositions, formulations, methods, and processes described herein include all actual or potential combinations of embodiments, aspects, options, examples, and preferences herein described. The exemplary compositions and formulations described herein may omit any component, substitute any component disclosed herein, or include any component disclosed elsewhere herein. The ratios of the mass of any component of any of the compositions or formulations disclosed herein to the mass of any other component in the formulation or to the total mass of the other components in the formulation are hereby disclosed as if they were expressly disclosed. Should the meaning of any terms in any of the patents or publications incorporated by reference conflict with the meaning of the terms used in this disclosure, the meanings of the terms or phrases in this disclosure are controlling. Furthermore, the foregoing discussion discloses and describes merely exemplary embodiments. All patents and publications cited herein are incorporated by reference herein for the specific teachings thereof.
Various embodiments and aspects of the inventions described herein are summarized by the following clauses:
(X-Z1-X-Z2-Z3-X-Z4-Z3)n,
(G-R-G-D-S-P-Y-S)m,
(X-Z1-X-Z2-Z3-X-Z4-Z3)n,
(X-Z1-X-Z2-Z3-X-Z4-Z3)n,
We have taken a different and complementary approach to understand how phase behavior is encoded in polypeptides. Analogous to—and inspired by—synthetic polymers that exhibit lower and upper critical solution temperature (LCST/UCST) phase behavior, we began by systematically scanning the sequence space of native IDPs to identify minimal peptide motifs that will confer LCST or UCST phase behavior when polymerized into a macromolecule that consists of many repeats of the peptide motif. With the greatly reduced sequence complexity of these repetitive polypeptides—compared to native IDPs that exhibit LCST/UST phase behavior—we then made rational changes in the amino acid repeat motif that systematically propagate along the sequence. These repetitive polypeptides can be rationally designed to exhibit both LCST and UCST phase behavior, and their phase behavior can be systematically modulated by amino acid mutations of the repeat motif. These artificial polypeptides also exhibit the same basic principles of phase separation inside cells as native IDPs.
Informed by a heuristic knowledge of factors that drive phase separation in repetitive polypeptides from these studies as well as the natural composition of membrane-less organelle IDPs, we set out to create artificial IDPs (A-IDPs) that exhibit phase separation in living cells to impart new functionality to the cell. Our design began with (G1-R2-G3-D4-S5-P6-Y7-S8)xx (where xx is the number of repeats between 20 and 80) a sequence inspired by Drosophila melanogaster Rec-1 Resilin, known to exhibit UCST phase behavior, that is chemically similar to IDPs that are critical constituents of membrane-less organelles (
We then used a subset of A-IDPs from this library to engineer intracellular condensates in living cells. The behavior of intracellular condensates for these A-IDPs proved to be surprisingly predictable and tunable, and enabled dynamic control over their cytoplasmic solubility and their interaction with the surrounding environment. Capitalizing on these observations, we created intracellular droplets capable of sequestering an enzyme whose catalytic efficiency within the engineered condensates can be genetically encoded by modulating the MW of the A-IDP.
pET24+ vectors were purchased from Novagen (Madison, Wis.). gBlock fragments encoding repetitive IDP (A-IDP) sequences of interest, superfolder GFP (sfGFP), mRuby3 and primers for pcDNA5 vector were purchased from Integrated DNA Technologies (Coralville, Iowa). Ligation enzymes, restriction enzymes, DNA ladders were purchased from New England Biolabs (Ipswich, Mass.). BL21(DE3) chemically competent Escherichia coli (E. coli) cells were purchased from Bioline (Taunton, Mass.). All E. coli cultures were grown in Terrific Broth media purchased from VWR Intemational (Radnor, Pa.). Kanamycin sulfate was purchased from EMD Millipore (Billerica, Mass.). Protein expression was induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) from Gold Biotechnology (St. Louis, Mo.). All salts, 10/40 kDa fluorescein labeled dextran molecules, L-(+)-Arabinose, L-Rhamnose and Fluorescein di(β-D-galactopyranoside) were purchased from Sigma-Aldrich (St. Louis, Mo.). 1× phosphate buffered saline (PBS) tablets (10 mM phosphate buffer, 140 mM NaCl, 3 mM KCl, pH 7.4 at 25° C.) were purchased from EMD Millipore (Billerica, Mass.). KRX E. coli cell line that endogenously expresses mutated LacZ were purchased from Promega (Madison, Wis.). NHS Ester reactive fluorophores (NHS-Alexa Fluor® 350 and NHS-Alexa Fluor® 647) were purchased from Life Technologies (Grand Island, N.Y.). DNA extraction kits, DNA gel purification kits were purchased from Qiagen Inc. (Germantown, Md.). Expi293 Eurkaryotic Expression System for HEK293 expression was purchased from Thermo Fischer Scientific (Waltham, Mass.). Whatman Anotop sterile syringe filters (0.02 μm) were purchased from GE Healthcare Life Sciences (Pittsburgh, Pa.). ABIL® EM 90 and TEGOSOFT® DEC surfactants were purchased from Evonik Industries (Essen, Germany). A single emulsion droplet-generating chip was purchased from Dolomite Microfluidics (Royston, United Kingdom). Syringe pumps were acquired from Chemyx Inc. (Stafford, Tex.).
A search of the literature provided an excellent list of intrinsically disordered proteins or protein regions are present in genes known to form membrane-less organelles. Each gene was divided into disordered regions and ordered regions according to the Predictor of Natural Disordered Regions (PONDR) VSL2 algorithm which is a meta-predictor of protein disorder of various lengths. Amino acid quantity was normalized to total protein length.
Each octapeptide amino acid motif inspired by our proteomic analysis was propagated twenty times in silico. This repetitive amino acid sequence was fed into an algorithm that creates an optimally non-repetitive DNA template from a repetitive protein gene. This 20-mer repeat gene was then ordered from IDT with Gibson assembly overhangs for easy insertion into modified pET24+ vector. To increase the number of total repeats of the gene, we performed iterative cloning steps of Recursive Directional Ligation by Plasmid Reconstruction adding an addition twenty repeats during each step. Transformations were performed into the desired E. coli cell line—BL21(DE3) for recombinant expression and single plasmid confocal experiments and a modified BL21(DE3) cell line termed KRX by Promega that contains a mutated LacZ gene for enzymatic experimentation.
In experiments with dual expression, genes were inserted into the pBAD33.1 vector by cutting custom pET24+ vector and pBAD33.1 cut with Hind III and Xba I. Gel purification was used to isolate the gene of interest from the housing pET24+ vector, which was then ligated into the similarly cut pBAD33.1 vector. Co-transformation was performed with ˜1 ng final concentration of each plasmid on kanamycin/chloramphenicol dual selection plates.
Individual liquid cultures of BL21 E. coli strains each harboring our gene of interest from Table 2 or Table 3 were inoculated into 5 mL of Terrific Broth (TB) medium from frozen glycerol stocks and grown to confluence overnight (16-18 hours). Cultures were then inoculated at a 1:200 dilution in 1 L TB media supplemented with 45 μg mL−1 kanamycin. Cells were grown at 37° C. in a shaking incubator (˜200 RPM) for 9 h, at which time protein expression was induced by the addition of 500 μM IPTG (final concentration). Cells were then incubated at 37° C. (shaking at ˜200 RPM.) for an additional 18 h. Protein was then purified from the insoluble cell suspension fraction. In brief, cell pellets were isolated by centrifuging cultures at 3500 RCF and resuspending in 20 mL of milli-Q water. Cells were then lysed by sonicating the cell solutions for 2 minutes, with 10 seconds of pulsing followed by 40 seconds of rest on ice (Misonix; Farmingdale, N.Y.).
Centrifuging each lysate suspension at 20,000 RCF for 20 minutes results in a soluble and insoluble fraction. The supernatant was discarded with the insoluble fraction resuspended in an approximately equal volume of 8 M urea+150 mM PBS (˜6-8 mL). For proteins with a fluorescent fusion tag, the insoluble fraction was resuspended in 3× insoluble volume at a final concentration of urea of ˜2 M to prevent protein misfolding. This suspension was heated for 10 min in a 37° C. water bath and then centrifuged at 20,000 RCF for 20 minutes. The supernatant was collected from this suspension and dialyzed in a 10 kDa membrane (SnakeSkin™, Thermo Fischer Scientific) against a 1:200 milli-Q water solution at 4° C. The dialysis water was changed twice over a 48-hour period. From inside the dialysis bag, both insoluble and soluble components were collected and centrifuged at 3500 RCF for 10 minutes and 4° C. The supernatant was removed and the remaining insoluble pellet containing the protein of interest was lyophilized for a minimum of three days to remove all water from the pellet.
Protein purity was characterized by 4-20% gradient tris-HCl (Biorad, Hercules, Calif.) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and staining with either 0.5 M copper chloride or SimplyBlue™ SafeStain (Thermo Fischer Scientific). Protein yield was determined by weight after lyophilization.
Creation of Water in Oil Droplets with Chip Microfluidics
To create water-in-oil emulsion droplets, two liquid phases—a dispersed, aqueous phase containing protein of interest in 150 mM PBS and an organic, continuous phase comprised of 75%/5%/20% vol/vol TEGOSOFT® DEC/ABIL® EM 90/mineral oil—were injected into the microfluidic droplet generators at constant flow rates using precision syringe pumps. The flow rates of the dispersed and continuous fluids were tuned to ensure droplet formation in the dripping regime; in these experiments, the dripping regime was achieved using a constant flow rate of 500 μL hr−1 for the organic continuous phase and 50-75 μL hr−1 for the aqueous, dispersed phase. The production of droplets within the microfluidic device was monitored using a 5× objective on an inverted microscope (Leica) equipped with a digital microscopy camera (Lumenera Infinity 3-1 CCD).
Circular Dichroism (CD) spectroscopy was performed using an Aviv Model 202 instrument and a 1 mm quartz sample cell (Hellma). A-IDPs were prepared by dissolving the purified lyophilized product in 5 mM PBS, pH 7.4 at a final concentration of 10 μM. The CD spectra were obtained at 50° C. from 260 nm to 180 nm in 1 nm steps at a 0.5 second average time. Data points with a dynode voltage above 500 V were ignored in the analysis. The CD spectra were corrected for the 5 mM PBS buffer signal at 50° C. This data collection was repeated in triplicate, and the average of the three measurements was represented as molar ellipticity.
Dynamic light scattering (DLS) measurements were performed over a temperature range of 10-80° C. using a Wyatt DynaPro temperature-controlled microsampler (Wyatt Technology, Santa Barbara, Calif.). Samples for the DLS system were prepared in 1×PBS and filtered through 0.02 μm Whatman Anotop sterile syringe filters (GE Healthcare Life Sciences, Pittsburgh, Pa.) into a 12 μL quartz crystal cuvette (Wyatt Technology, Santa Barbara, Calif.). 5 acquisitions were taken at each temperature for a 5 second duration, and the results presented represent the mean Rh of the sample at each temperature.
Cloud point transition temperatures (Tt) were determined via temperature-controlled spectrophotometry using a Cary 300 (Agilent Technologies). Samples containing various concentrations of protein in 150 mM PBS were cooled at 1° C./min while the absorbance at λ=350 nm was recorded every 1° C. Absorbance was normalized to the absorbance at the highest temperature point collected, corresponding to the more soluble point during a given experiment. The cloud point was determined as the maximum in the first derivative of the absorbance as a function of temperature. Transition temperature was calculated by the point of minimum slope. Saturation concentration was defined by the natural logarithm fit line created from a minimum of three volume fractions. Error bars are standard error of the mean from three repeats of a minimum of three transition temperatures.
The uptake of dextran molecules into the phase separated space of [WT]-20 and [Q5,8]-20 was performed to quantify the isolation of A-IDPs from their surroundings. Fluorescein isothiocyanate labeled dextrans (10 kDa, 40 kDa, Sigma-Aldrich, St. Louis, Mo.) were added to 4 mg mL−1 solutions of unlabelled [WT]-20 and [Q5,8]-20 at final concentrations of 4 mg mL−1 and 1 mg mL−1, respectively at 60° C. Soluble samples were then transferred to room temperature glass slides and mounted with #1.5 cover slip. Samples were imaged on an upright Zeiss Axio Imager D2 microscope with a 20× objective and the appropriate filter set (ex 470/40, em 525/50) after 1-hour incubation below the transition temperature. Fluorescent intensity was calculated from background corrected fluorescent intensities inside/outside droplets in ImageJ portioned using bright field images of the phase separated space.
Sample Preparation for Temperature Gradient Experiments High concentration A-IDP stock solutions (60 wt %) were prepared by resuspending a mass of lyophilized A-IDP pellets with an appropriate volume of phosphate buffer saline solution (PBS) at a solution pH of 7.0. The concentration was converted to mg mL−1 by assuming that the density of the A-IDP was 1 g mL−1. The RLP stock solution was heated in a water bath at 85° C. for 60 minutes and mixed periodically along with sonication to ensure homogeneity. Lower concentration samples were made by mixing the initial stock solution volumetrically with PBS at a pH of 7. To prepare for temperature gradient microfluidics (TGM) measurements, the solutions were loaded into 12 mm×1 mm×0.1 mm rectangular borosilicate glass capillary tubes (VitroCom, Inc.), by capillary action, and sealed with wax to avoid sample evaporation and convection. The capillary tubes were held in contact with a hot plate at 85° C. housed within an incubator at 65° C. during the loading process. The high temperature environment ensured that the RLP solutions were held above the critical phase transition temperature (˜85° C. for [WT]-20). Capillary arrays were prepared by taping several capillaries together. The arrays were stored at 85° C. in an oven for 10 minutes prior to subjecting them to the temperature gradient experimentation.
The temperature gradient device imposed a linear temperature gradient across the A-IDP solutions. This was accomplished by placing the glass capillary array into thermal contact with a heat source on one side and a cold sink on the other. The sample was then bathed in white light. This light was scattered by phase separated A-IDP droplets at cold temperature and was imaged via dark-field microscopy. The temperature gradient was calibrated for each experiment using two reference solutions placed alongside the A-IDP samples of interest. The cold temperature calibration reference contained 10 mg mL−1 poly(N-isopropyl acrylamide) (PNIPAM) with MW=1.868×105 g mol−1 in H2O (Polymer Source, Inc.). The hot temperature calibration reference contained 10 mg mL−1 poly(ethylene oxide) (PEO) with MW=9×105 g mol−1 in a 1 M NaCl aqueous solution (Sigma-Aldrich). The LCST of each reference solution was obtained with a melting point apparatus that measured the light scattering intensity as the temperature was increased at a rate of 0.5 K min−1. When placed onto the temperature gradient device, the reference solutions became cloudy at temperatures above the LCST. The pixel position of the LCST was obtained by the onset of light scattering intensity relative to the low intensity baseline on the cold side of the capillary. The temperature gradient was calculated using the pixel positions and the LCSTs of the two samples, assuming a linear relationship between position and temperature.
Fits for the roughly dilute, overlap and semi-dilute regions of our obtained phase diagrams were calculated using fitting methods for lower critical solution transition polypeptides adopted for upper critical solution transition polypeptides as described previously.
Briefly, for low volume fractions (ϕ<0.1), A-IDPs exhibit roughly a log-normal dependence on UCST cloud point with respect to volume fraction as seen with other repeat polypeptides. For the high density regime (ϕ>˜0.4) using surface tension scaling methods previously described for elastin-like polypeptides, we determined the coefficients of proportionality (A) and estimated theta temperature (θ) of [WT]-20 and [Q5,8]-20 to be A=−0.00092, θ=389 K and A=−0.00092, θ=392 K respectively. In a poor solvent, the surface tension of a dilute phase globule γ can be written in the form γ≈C kTb2 ϕ″2 where b is the polypeptide Kuhn length (b=2.2 nm as measured by Fluegel and co-workers for other repetitive polypeptides) and C an adjustable coefficient. Replacing the surface tension γ by kTϕ2{circumflex over ( )}2*A*(T−θ) we obtain an equation for the temperature dependence of the coacervate volume fraction
Using a least-squares fit in Igor (WaveMetrics Inc. Portland, Oreg.), we adjust the coefficient C for this temperature dependence to match the measured [WT]-20 binodal and the [Q5,8]-20 binodal points to determine C=0.62 and 1.05, respectively.
Closer to the critical point in the so-called Ginzburg zone one needs to use the critical Ising model to describe the phase behavior of polymer solutions. The phase boundary in the critical zone varies more gradually than predicted by mean field theory:
where Tc=351.5 K for [WT]-20 and Tc=332.3 K for [Q5,8]-20, 0.3 is the critical Ising exponent (Flory-Huggins mean field value is 0.5) for both [WT]-20 and [Q5,8]-20, and Cc is the fitting coefficient. We calculated fitting coefficients in Igor (WaveMetrics Inc. Portland, Oreg.) equal to 1.29 and 1.27 for [WT]-20 and [Q5,8]-20 respectively. Note that we calculated ϕ1 explicitly using data collected with UV-Vis spectrophotometry and according to natural logarithm fits described in Table 1.
Cells were grown overnight in 5 mL of TB media from glycerol stocks. In conjunction to fluorescent or confocal imaging, cells were analyzed for total sfGFP fluorescence and OD600. Briefly, 50 ul of cell culture at various time points was resuspended in 1 mL of 150 mM PBS. Using a combination of a UV-Vis spectrophotometry signal from a NanoDrop 1000 (Thermo Fisher Scientific, Waltham, Mass.) and fluorescent spectra from a NanoDrop 3300 (Thermo Fisher Scientific, Waltham, Mass.), we calculated the relative ratio of sfGFP fluorescence normalized to cell density. Using this information in conjunction with imaging analysis, we were able to determine the intracellular saturation concentration normalized to cell density.
Temperature Controlled Fluorescent Microscopy of Protocell Droplets and E. coli Bacteria
Water-in-oil droplets were collected on a glass microscope slide and cooled using a precision Peltier heating and cooling stage (Linkam LTS120) equipped with a temperature control unit (Linkam PE95). The spatial distribution of Alexa Fluor 350-labeled (25% molar fraction N-terminal labeled) [Q5,8]-20 and Alexa Fluor 594-labeled +4 Net was characterized via fluorescence microscopy using an upright Zeiss Axio Imager D2 microscope with a 20× objective and the appropriate filter set. Similarly, intracellular pattering of A-IDP-superfolder GFP over time was characterized via fluorescence microscopy using an upright Zeiss Axio Imager D2 microscope with a 20× objective and the appropriate filter set (ex 470/40, em 525/50). Cell fluorescent was calculated using ImageJ software. Temperature ramps began at various temperatures but always were set to a constant speed of 5° C./min.
[WT]-20-sfGFP was extracted from the pET24(+) vector using polymerase chain reaction (PCR). Briefly, the forward and reverse primers were resuspended with 1 ng of pET24(+) plasmid containing [WT]-20-sfGFP gene fusion. Using a PCR cycle of [98° C., 1 min; 65° C., 30 sec; 72° C., 2 min]×30 cycles, followed by gel purification, the gene was finally constructed with Gibson assembly. pcDNA5 vector containing [WT]-20-sfGFP was transfected into HEK293 cells according to manufacturer instructions (Expi293 Expression System, Thermo Fischer Scientific, Waltham, Mass.). Cells were spun down at 500 RCF for 10 min at room temperature on day 5 of transient transfection and resuspended in 150 mM PBS for imaging.
Cells were prepared as follows. A tube containing 5 mL of TB media was inoculated overnight with protein of choice from bacterial glycerol stock. After 16 hr of growth, induction with 1 mM IPTG and 2% L-rhamnose (Sigma-Aldrich, St. Louis, Mo.) was added each flask of interest. Samples were collected at the indicated time points and prepared for imaging as follows: 50 μL of cell suspension was pelleted under 20,000 RCF for 1 min at room temperature. Cells were resuspended to OD600=0.15 at 1 cm path length. 50 μL of resuspended bacterial cells were transferred to a 384-well plated with #1.5 glass bottom (Cellvis). There was a 10 min equilibration period to the incubation chamber prior to each time point data collection.
Images were collected at different time points with a 63× oil-immersion objective on a Zeiss 710 inverted confocal with temperature-controlled incubation (Car Zeiss AG, Oberkochen, Germany). sfGFP fluorescent was detected with a 488 nm excitation laser and 488/594 emission filter. Data was primarily taken at 25° C. unless otherwise noted. All fluorescent quantification and cell portioning analysis was performed in ImageJ.
In colocalization experiments, cells were grown overnight from glycerol stock in dual antibiotic media containing 45 ug/mL kanamycin and 25 μg/mL chloramphenicol (final concentration). After 16-18 hours, pET24(+) expression was induced with 1 mM concentration of IPTG (final concentration). After 24 hours of IPTG induction, media was replaced with 5 mL of TB supplemented with 1 mM IPTG and 2% arabinose (final concentration) (Sigma Aldrich, St. Louis, Mo.). After 9 hours of induction with both, cells were prepared for confocal imaging by spinning down 50 μL of culture at room temperature and resuspended in 150 mM PBS to OD600=0.15 at 1 cm path length. All imaging details remain the same except that mNeonGreen/sfGFP detection was performed with 488 nm excitation laser and 488/594 emission filter and mRuby3 detection with 561 nm excitation laser and 488/561 emission filter.
Cells were prepared as follows. A tube containing 5 mL of TB media was inoculated overnight with protein of choice from bacterial glycerol stock. After 16 hr of growth, induction with 1 mM IPTG and 2% L-rhamnose (Sigma-Aldrich, St. Louis, Mo.) was added each flask of interest. ˜24 hours later, 50 μL of cell suspension was pelleted under 20,000 RCF for 1 min at room temperature. Cells were resuspended in 150 mM PBS to OD600=0.15 at 1 cm path length. Fifty microliters of sample were added to Culture-Insert 4 Well (1.5 coverslip, Ibidi, Madison, Wis.) petri dishes and allowed to incubate at room temperature for 10 minutes. After incubation, 2 μL of 1 mg mL−1 FDG resuspended in 98% water, 1% DMSO and 1% EtOH was added. Imaging began immediately (within 20 seconds) and images were captured every minute for 30 minutes total. Imaging was performed on an Andor Dragonfly Spinning Disk 500 series confocal on a LeicaDMi8 microscope stand (Oxford Instruments, Abingdon, UK) with a 63× water immersion objective and equipped with a Zyla 4.2 series camera. Converted FDG was detected with a 488 nm excitation laser and 525/50 nm emission filter and mRuby3 fluorescence with a 561 nm excitation laser and 600/50 nm emission filter.
Fluorescent Spectroscopy for Determining Km, Vmax and kcat
Liquid cultures of KRX E. coli containing plasmid of interest were grown from glycerol stocks overnight (16-18 hours). Cells were then induced with 1 mM IPTG and 2% L-rhamnose (Sigma-Aldrich, St. Louis, Mo.) for 24 hours. Cells were pelleted and resuspended at OD600=˜0.15 in 140 mM PBS. Various concentrations of FDG were added while monitoring fluorescent intensity at 520 nm using NanoDrop 3300 (Thermo Fisher Scientific, Waltham, Mass.). The same instrument was used to also calculate the fluorescent intensity of mRuby3 as a relativistic measure of expression level of the various alpha peptide fusions. Plotting the observed fluorescent intensity at different times provides a surrogate measure of the rate of hydrolysis at various concentrations of the substrate (Vo). These rates were then converted into typical Lineweaver-Burk conventions to determine Vmax and Km. For consistency in units, [FDG] was converted into fluorescent intensity using a fluorescein standard curve of y=185919*[FDG in mg]+1045. This conversion assumes that converted FDG into fluorescein has similar fluorescent intensity profile to free fluorescein dye.
For experiments performed with regard to determining the intracellular fluorescent intensity of A-IDP-sfGFP at various points post-IPTG induction the following statistical analysis was performed. For determining the saturation concentration intracellular, whole cell fluorescence normalized to cellular density (OD600) on three independent samples was calculated while imaging of their intracellular architecture. Upon first observation of phase separation in E. coli in more than 50% of cells within a microscopic field of view, this normalized cell density was recorded as the saturation concentration. Data is normalized to data collected for [WT]-40 as a reference point. Error bars represent propagated standard error of the mean of three separate samples from the same original cell suspension.
With the microscope images collected with confocal microscopy at various time points, we isolated the soluble and puncta fractions within the cells at various points in time via analysis in ImageJ. Puncta consistently create pixels dense enough to saturate the detector while simultaneously observing the rest of the cell. Thus, by thresholding around the upper 2% of total pixel intensities, one can easily partition this section from the remaining cell cytoplasm. Using this constant thresholding between timepoints in each experimental group, we were able to track the total size of these puncta over time with regard to the total size of the cell (puncta+soluble fraction). Error bars of these data are standard errors of the mean of normalized puncta (two-phase) area of three images of different fields of view of the sample overall cell samples. These two channels are combined and split differently in
Given the lack of automated tools for the detection of intracellular phase separation between two images, we calculated the intracellular transition temperatures manually. Similar to the detection of phase separation with UV-Vis spectrophotometry, the intracellular transition temperature was determined as the midpoint between a frame that was certainly homogenous and a second frame that was certainly two phases. All transition temperatures were determined in this way, going from a point of solubility to insolubility whether the solution was being heated or cooled. Due to the level of subjectivity of this assessment, sample identifiers were blinded to the analyst and a high number of cells were analyzed in each experiment (n=30). Data was normalized to the initial mean fluorescence of the homogeneous cells at a consistent temperature (often 60° C. unless otherwise noted). Error bars indicate standard error of the mean.
The error bars of dextran fluorescence indicate the standard error of the mean fluorescence inside and outside of the phase separated space from three separate fields of view.
For quantification of Fluorescein Di-β-D-Galactopyranoside (FDG) relative to the different expression levels of alpha peptide, channels were split between fluorescence from FDG and mRuby3 respectively. Using the particle analysis tool from ImageJ, areas of green fluorescence were isolated from the background. If the mean fluorescence of this area was 5% greater than the background fluorescence (mean fluorescent of the area excluded by the previous particle mask), then this particular particle's background subtracted green fluorescence was included in the analysis. Particles were excluded if their area was below 0.1 um2. Using the same particle mask, the background subtracted mean fluorescence of mRuby3 was calculated on the other fluorescent channel. We report the ratio of these two channels as a surrogate for enzymatic efficiency. Error bars are standard errors of the mean at each timepoint.
For quantification of Fluorescein Di-β-D-Galactopyranoside (FDG) inside the cellular space versus outside the cellular space, channels were first split between fluorescence from FDG and mRuby3 respectively. Using the same particle analysis tool from ImageJ, areas of green fluorescence were isolated from the background. If the mean fluorescence of this area was 5% greater than the background fluorescence (mean fluorescent of the area excluded by the previous particle mask), then this particular particle's background subtracted green fluorescence was included in the analysis. Particles were excluded if their area was below 0.1 um2. Ratio of fluorescent intensity inside of cells versus the extracellular space is the background corrected mean fluorescence of FDG divided by the background fluorescence. Error bars are standard errors of the mean at each timepoint.
To quantify the amount of colocalization we used the Coloc2 plug-in available through ImageJ software. Using automated thresholding, we report the Mander's colocalization coefficient which accounts for the intensity to the two channels of interest as described previously.
Identification of a Minimal IDP Repeat from Proteomic Analysis and Sequence Heuristics
We conducted a proteomic analysis of 63 IDPs that form membrane-less organelles to investigate their sequence composition. We were particularly interested in categories of amino acids suspected to drive phase behavior via intrachain interactions, such as charge-charge, cation-π and hydrogen bonding via non-charged polar residues (
In order to manage the vast sequence space of all possible mutations of the octapeptide repeat, we classify each amino acid into categories of intrachain interactions that could contribute to UCST phase behavior. N, Q, S, T are classified as polar, uncharged amino acids. R-K and D-E are pairs of positively charged and negatively charged amino acids. G and P are placed into a separate category given their unusual structure and importance in promoting a disordered polypeptide backbone (
The wild-type (WT) repeat unit is (G-R2-G3-D4-S5-P6-Y7-S8)40 where 40 refers to the number of repeats. The MW of the A-IDPs was varied between ˜15 and ˜70 kDa—by varying the number of repeat motifs from 20 to 80—to account for observed differences in MW in the intrinsically disordered regions (IDRs) of naturally occurring IDPs (
One advantage of A-IDPs is their minimal interaction with other proteins or biomolecules stemming from their repetitive nature. This feature of A-IDPs combined with their reversible aqueous two-phase separation enables simple column-free purification by UCST phase transition cycling between the one- and two-phase regime of the phase diagram. An example of this purification process is shown in
A-IDPs [WT]-20 and [Q5,8]-20 exhibit UCST phase behavior in vitro. To characterize their phase transition behavior, we employed three different techniques. First, we utilized droplet microfluidics, where monodisperse water droplets are formed in oil containing the A-IDP of interest (
Second, we employed temperature dependent dynamic light scattering (DLS) to observe the two-phase separation in bulk. A solution of [Q5,8]-20 is heated to 80° C. and DLS data was collected as the solution is cooled to 10° C. We observe a transition from soluble A-IDP molecules with a hydrodynamic radius (Rh) of 4 nm to aggregates larger than 1 μm as a function of temperature (
Third, we employed temperature-dependent turbidity measurements at a fixed wavelength of 350 nm to characterize the UCST phase separation while heating and cooling a solution of an A-IDP at a rate of 1° C. min−1 (
To understand the effects of a particular residue substitution in the octapeptide repeat on the phase separation for the A-IDP, we created a set of “mutant” A-IDPs ranging from 100% of a to 100% of b where a is the WT repeat unit. The doping scheme wherein the mutant repeat unit b is periodically inserted into the WT sequence is visually illustrated by the color-coded schematic in
The Tt of the WT and each A-IDP is a linear function of its volume fraction (ϕ) (
We next tested the effect of fifteen different site-specific substitution mutations of the reference—WT—repeat motif on the saturation concentration (Csat)—defined as is the concentration at which the Tt is 37° C.—of the A-IDPs. We found that single residue changes in the octapeptide repeat are capable of changing the Csat—normalized to the degree of substitution defined by the percent change in amino acid composition—of the repeat polypeptide by over by two orders of magnitude at constant molecular weight that ranged from 1-800 μM (
These substitutions present quantitative evidence for the importance of interactions between R and aromatic residues in the repeat motif of the A-IDP. When Y7 is substituted, we observe dramatic shifts in the UCST cloud point at ϕ=10−3, from 66° C. to 123° C., 59° C. and ˜2° C. for W, F or H respectively (
We next looked at the effect of A-IDP MW on phase behavior, we chose A-IDPs with MWs between ˜17 kDa and ˜70 kDa, as this MW range covers 75% of the IDRs in our proteomic analysis of native IDPs (
In addition to composition, concentration (ϕ) and MW on Tt, there are several other parameters that have a measurable effect on UCST phase behavior but that do not eliminate UCST phase behavior under physiologically relevant conditions. Uncharged polar substitutions, the ratio of G/P, the syntax of the repeating polypeptide, solution salt content, pH (in the absence of H) and identity of the negatively charged amino acid (E vs. D) all result in smaller changes to the UCST binodal phase boundary than MW, volume fraction, aromatic:aliphatic amino acid ratio and R content (
Having observed that Csat and the binodal phase boundary in the dilute regime of the UCST phase diagram of A-IDPs can be modified drastically by amino acid substitution, we were interested in the factors that modulate the high concentration regime of the phase diagram of A-IDPs. Polypeptides [WT]-20 and [Q5,8]-20 express extraordinarily well for recombinant proteins, with yields of ˜500 mg L−1 in shaker flask culture, which made it easy to purify over one gram of material to measure the UCST cloud point behavior at high-volume fractions of these A-IDPs directly (>0.1). To minimize the amount of material required, these experiments were performed in a multiplexed linear temperature gradient microfluidic device mounted on an upright light microscope, wherein the Tt could be quantified by the temperature at which phase separation occurs by a visible increase in light scattering intensity. These experiments produce binodal phase boundaries similar to optical turbidity measurements that are typically carried out in a UV-vis spectrophotometer (
A-IDPs have Controlled Csat in Eukaryotic and Prokaryotic Cell Lines
With a set of A-IDPs that exhibit a range of Tt as a function of concentration, and Csat that vary over seven orders of magnitude we sought to understand: (1) the dynamics of droplet assembly in living cells, and (2) to elucidate if it proceeds in vivo similarly to in vitro. To explore these two issues, we chose a set of IDPs that have a range of Csat from 1 to 815 μM with MWs of either ˜17 kDa or ˜32 kDa. To visualize localization of the A-IDPs within bacterial cells, each A-IDP was genetically fused to a super folder version of green fluorescent protein (sfGFP) (
Fusion of sfGFP to A-IDPs to [WT]-20, [WT]-40, [3Y7:V7]-40, [Y7:V7]-40 did not eliminate the phase behavior but shifted the phase diagram (
In contrast, phase separation in E. coli is significantly different from eukaryotic cells. The initiation of the UCST phase transition in E. coli is similar to HEK cells—and in vitro—where small densely fluorescent puncta form after the A-IDP concentration in the cell exceeds Cm, that then grow in size over time (
Similar to in vitro, the MW and aromatic:aliphatic content affect droplet formation in E. coli. Doubling the MW of [WT]-20-sfGFP to [WT]-40-sfGFP decreases Csat enough to cause droplet formation even prior to A-IDP induction, presumably because of leaky transcriptional regulation (
A-IDPs Exhibit Reversible UCST Droplet Formation in E. coli
Just as one can cross a binodal line into the two-phase regime under isothermal conditions by increasing polypeptide volume fraction, this line may be crossed under constant volume fractions by decreasing solvent quality or the chi parameter (X). Experimentally this is most easily accomplished by reducing the temperature of the bulk solution. Similar to the UCST phase behavior of A-IDPs in vitro, A-IDPs exhibit reversible UCST phase separation inside cells that is reversible by repeated four cooling and heating cycles (
Interestingly, upon multiple heating and cooling cycles, we observed that E. coli exhibit spatial phase separation memory, with puncta forming in the same location as the first cycle (
Increasing the MW of the A-IDP increases the observed Tt in E. coli (
In order to understand the potential of using spatially confined intracellular coacervate droplets to carry out new functions, we asked the following questions: (1) Can coacervate droplets in cells recruit other molecules, and if so, what, if any, are the size limitations of such molecules?(2) Can these molecules interact with the A-IDP to impart a new function to the droplet?
To answer these questions, we first examined whether a small molecule could diffuse into and react with the A-IDP in a coacervate droplet located within an E. coli cell. We designed and expressed an A-IDP—[3Y7:V]-40-UAA—that carries three copies of a unique biorthogonal reactive group—an azide; its primary amino acid sequence is listed in Table 3. After reaching intracellular concentrations greater than the Csat of [3Y7:V7]-40-UAA, we incubated live E. coli with 1 mg mL−1 dibenzocyclooctyne-dye (DBCO-Alexa488) for 10 min (
Next, we asked if larger molecules such as proteins are also capable of interacting with an A-IDP puncta. To answer this question, we designed a droplet capture experiment based on split green fluorescent protein (GFP). We first verified if the two components of a split GFP can interact with each other to create a functional GFP molecule if one of the components is fused to an A-IDP. GFP-11-[3Y7:V7]-40-mRuby3 was co-expressed in the presence of GFP-1-10; because the IDP is fused to mRuby3, the A-IDP condensates fluoresce red and can be visualized by fluorescence microscopy within the cell. We see fluorescently active GFP only in the interior of the condensates as seen by the co-localization of green fluorescence with the red fluorescence from the A-IDP condensates, indicating that the fragments GFP bind to each other to create an intact and functional GFP molecules that fluoresces green (
These results show that two protein fragments of GFP can find and bind to each other in the cell despite the steric hindrance imposed by an A-IDP and a fluorescent reporter fused to one fragment of the protein. It does not however, prove that a protein can be recruited after an A-IDP condensate has formed, as the protein partners in the previous experiment are co-expressed and could bind in the cytoplasm prior to phase separation that occurs once the intracellular concentration of GFP-11-[3Y7:V7]-40-mRuby3 exceeds its Csat. To directly answer this question, we co-transformed E. coli with two plasmids—a Lac operon regulated plasmid that encodes one fragment of GFP (GFP-11) that is fused to [3Y7:V7]-40 and a second plasmid regulated by araBAD operon that encodes the other fragment of GFP (GFP-1-10). Once expression of GFP-11-[3Y7:V7]-40 at 37° C. proceeds long enough that its intracellular concentrations is greater than its Csat. we removed the IPTG induction media, and replaced it with arabinose containing media that induce the expression of the larger GFP fragment (GFP-1-10). We observed that subsequent to arabinose induction, both the ϕ1 and ϕ2 fractions of the E. coli contained fluorescently active GFP (
These experiments clearly show that small molecules and proteins can be recruited into intracellular coacervate droplets in E. coli and that a protein can be reconstituted within a coacervate droplet. These results suggested a path for the de novo design of intracellular coacervate droplets with new enzymatic function. We chose biocatalysis as the function of interest, because one of the proposed reasons for the evolutionary development of biomolecular condensates is to modulate the kinetics of various biological functions, including enzymatic reactions. However, there is little experimental evidence demonstrating how the function of enzymes is modulated by phase separation.
To investigate this, we created an A-IDP fusion that can recruit an enzyme into intracellular droplets to modulate its catalytic activity. We chose β-galactosidase for two reasons: (1) it has a range of small molecule substrates, one of which, Fluorescein Di β-Galactopyranoside (FDG), is colorless but when cleaved by ß-galactosidase, will fluoresce green. Thus, using a combination of a red fluorescent protein tagged to our enzyme-A-IDP fusion and fluorescein florescence we can track the colocalization of enzymatic reactions with A-IDPs in real time. (2) We had concerns that a large enzyme fused to a large A-IDP would not express at high enough concentrations in E. coli and thus not phase separate in vivo. To alleviate this concern, we took advantage of the widely used β-galactosidase (LacZ) blue-white screening system, where the alpha peptide (αp) complements the mutated enzyme LacZΔM15 to create a functional β-galactosidase enzyme. In our system, the αp is fused to a A-IDP-mRuby3 construct, such that enzyme activity is physically linked to the A-IDP which in turn is physically linked to red fluorescence.
Our studies with the DBCO-Alexa488 and split GFP provided the basis for this more complicated experiment. The DBCO-Alexa488 experiment suggested that a small molecule such as an enzyme substrate can penetrate puncta, even if delivered extracellularly (
Thus, we genetically fused the α-peptide (αp) from LacZ β-galactosidase to a A-IDP-mRuby3 construct. Our hypothesis is that the αp-A-IDP-mRuby3 protein can bind and recruit the other fragment of the enzyme—LacZΔM15 that has an α-peptide deletion—that is expressed endogenously in genetically modified E. coli (KRX, Promega) into intracellular droplets. After protein induction and resulting condensate formation, we deliver the substrate Fluorescein Di β-Galactopyranoside (FDG) to the cell medium where it is trafficked intracellularly, hydrolyzed into fluorescein at the sites of active β-galactosidase, and eventually exported outside the cell (
In our control experiment—αp-mRuby3—we observe limited persistence of fluorescence within the cells. It is important to note that the α-peptide itself is known to form inclusion bodies, and therefore, even in this control experiment, we observe some puncta inside the bacterial cells. However, upon fusion with [WT]-20, we observe that the fluorescence localizes long enough with the A-IDP condensates to be observed with confocal microscopy (
When we increase the MW of the A-IDP, and thus decrease Csat, we observe a dose-response effect in the total FDG fluorescence intensity as well as colocalization with the αp-A-IDP-mRuby3 fusion (
Quantification of fluorescence production at various substrate concentrations in vitro suggests that the mechanism of this effect is a statistically significant increase in the catalytic constant (Kcat) of the enzyme with increasing MW. This constant can be interpreted as the “turnover efficiency” of the enzyme or the number of catalytic events that occur per unit time. We observed 1.4×, 1.6× and 4.2× increase in the Kw for αp-[WT]-20-mRuby3, αp-[WT]-40-mRuby3, αp-[WT]-80-mRuby3 compared to the αp-mRuby3 control (
We also fused the LacZ alpha peptide to A-IDPs with differing levels of aromatic content at a constant MW (
We show herein A-IDPs that consist of repeats of an octapeptide motif inspired by native IDP exhibit reversible UCST phase separation in aqueous solution. Despite the simplicity of their sequence, they recapitulate many of the features seen in more complex, native IDPs. The formation and dynamics of their phase separation into coacervate droplets are controlled by two simple design parameters that are genetically encodable at the sequence level—MW of the A-IDP and the ratio of aromatic:aliphatic residues in the octapeptide repeat. Using these two parameters—aromatic:aliphatic ratio and MW—we were able to produce A-IDPs with Csats ranging from nanomolar to millimolar concentrations. This work supports the growing evidence of R-aromatic interactions that drive phase behavior and also adds additional evidence of the molecular hierarchy that exists between the aromatic groups W, Y, F and H in modulating UCST phase behavior. Although the IDP literature often ignores the importance of MW, our results suggest that MW may be more critical than composition for defining the UCST binodal. We anticipate that these results will inform and dramatically shift the strategy for mutating native IDPs and designing de novo IDPs.
These design parameters faithfully translate from in vitro to intracellular environments. The A-IDPs phase separate inside cells by the same principles that drive their UCST phase separation in vitro indicating that the same thermodynamic driving forces embedded in the sequence and molecular weight also modulate droplet formation dynamics in isolation. Due to the simplicity of their design, A-IDPs behave in vivo as their phase diagrams in vitro suggest—as their intracellular concentration increases to a Csat, small phase separating droplets form at individual points in space that continue to grow in size with increasing overall A-IDP concentration inside the cell. This predictable observation has been theorized by previous studies but has not been conclusively demonstrated until now.
Finally, these proteins can be used for the de novo design of functional intracellular droplets. We rationally designed intracellular puncta capable of binding and recruiting a β-galactosidase deletion mutant, which could modify the catalytic efficiency of the enzyme-substrate complex—a complex which has not evolved to form intracellular condensates. The catalytic efficiency of the reconstituted enzyme in phase separated coacervate droplets is MW dependent and increases with the MW of the A-IDP. Higher MW A-IDPs more efficiently sequester the substrate in the enzymatically active, intracellular phase separated puncta, which results in a higher catalytic efficiency as measured by Kcat. These proof-of-concept experiments demonstrate that intracellular droplets can be engineered to have non-canonical functions in live cells and provide a new platform for intracellular material manipulation. In summary, with over 60 IDPs synthesized for this study that span a range of Csat, and proof of concept experiments recruiting proteins into coacervate droplets within a cell and thereby modulating protein function, these studies lay the groundwork for the de novo design of functional intracellular condensates. We expect that these A-IDPs will be useful as building blocks from which new biological condensates with emergent behaviors can be built within living cells to better study the functional significance of phase separation in living cells and to encode new functions for droplets within cells. We also anticipate that these IDPs will prove useful in other biomedical applications beyond the design of intracellular droplets that can profit from the their tunable UCST phase behavior. This marriage of soft material science with biophysical characterization of subcellular materials will continue to be an exciting space for to engineer cells with new or improved function and new biomaterials.
Table 8 shows the expression levels of various fusion proteins.
Effect of Repetitive Polypeptide Design on In Vivo Release of Glucagon Like Peptide 1 (GLP-1) from Sub-Cutaneous Depots
We utilized the phase behavior of the repeat polypeptide, chemically inspired from naturally occurring IDPs, to control the bio-availability of resources when confined by a lipid bi-layer in bacteria and to control the assembly of micelles when sterically confined by a relatively hydrophilic ELP or RLP molecule exerting a surfactant like effect. We were motivated to test the efficacy of controlling bio-availability in a system where the dilute phase is attached to an infinite sink—where the dilute phase is in equilibrium with a biological system capable of protein clearance. The Chilkoti lab has extensive experience with subcutaneous delivery of peptide molecules in vertebrae animals in this exact set up, the primary focus on delivering therapeutic molecules for diabetes and various cancers, albeit exclusively with LCST polypeptides.
The delivery of peptides remains an outstanding challenge for drug delivery. Despite protein engineering improvements focused on improving half-life, their effective window lasts from minutes to a few hours rendering them unsuitable for therapeutic use. Interestingly, nature utilizes peptides in various biological applications, but regulation of their activity is often tightly controlled by a cellular population that can react to a phenotype change. Thus, man-made peptide drugs require a delivery solution, one that can improve the pharmacokinetics of these valuable macromolecules.
The most common approaches to improve a peptide's half-life include protein engineering and changing the formulation to prolong release and/or reducing renal clearance. Sequence engineering—such as the incorporation of D-amino acids or other chemically esoteric amino acid derivatives—can limit proteolytic degradation of the protein, but severely limit manufacturing choices. Encapsulation methods produce inconsistent effects on bioavailability or require harsh production conditions that limit the type of peptide drug that can be delivered via these methods. Strategies to decrease renal clearance revolve around increasing the size of the molecule and reducing opsonization including attachment to synthetic or biological polymers that extend half-life, fusion to a large protein, and conjugating chemical moieties that allow the peptide to piggyback on endogenous biomolecules with slow turnover rates like albumin or antibody fragments. These strategies are not without limitations as they dramatically reduce potency and rely on a patient population to express these piggybacking biomarkers consistently between individuals.
These engineered polypeptides offer an elegant solution-through their primary amino acid sequence and molecular weight they control the dense and local dilute phase of the bioactive molecule effectively controlling the bioavailability of the drug. Unless proteases possess the unique ability to diffuse into the dense phase of the polypeptide-peptide drug depot, the availability of peptide drug is mediated exclusively by the primary amino acid sequence of the polypeptide. This local dilute phase can then diffuse from the subcutaneous space into circulation and exert a therapeutic effect. Using the principles of polypeptide design described herein we can rationally design drug release depots that can prolong the half-life of the peptide drug in vivo.
For this study, the relevant peptide drug is GLP-1, which is a 31 amino acid peptide produced in the L cells of the intestines, capable of exerting blood glucose control over a large therapeutic window. Previous experience with GLP-1-polypeptide depots revealed much about the design of sub-cutaneous depots for drug release. (1) Fusion of a macromolecule such as the polypeptide ELPs reduce potency of the GLP-1 molecule by about ˜30 fold but this does not preclude in vivo activity. (2) Zero order release can be achieved for up to 10 days in mice and 17 days in monkeys under optimal conditions. (3) Optimal conditions are an injectable transition temperature 5-7° C. below the body temperature of the animal and a molecular weight of 35 kDa or greater to avoid renal clearance. This optimal 5-7° C. below body temperature corresponds to a dilute phase concentration of approximately 1-100 μM where non-optimal depots exhibited Csats an order of magnitude above and below this optimal range.
The peptide drug of choice for these experiments is GLP-1 for several reasons. (1) GLP-1 can rapidly exert a therapeutic effect in vivo. (2) GLP-1 is a prime candidate for improved pharmacokinetics with a half-life of ˜5 min in vivo. (3) GLP-1 can be easily studied in established mice models of diet induced obesity where a high fat diet increases the blood glucose. 4) GLP-1 is a stable peptide drug which will eliminate confounding variables associated with genetic fusion to various polypeptide partners and myriad delivery strategies.
Previous studies suggested that transition temperature at injection concentration is the important parameter for determining efficacy. However, this misjudges the important isotherm on the phase diagram to be room temperature instead of the operating temperature of the depot which is defined by the animals resting body temperature. In subcutaneous mice models, this temperature is approximately 35° C. Thus, we are looking proteins with variable dilute phase concentrations, at an isotherm of 35° C. (similar to a saturation concentration at 35° C.) and different molecular weights to test to observe how these two variables affect the bioavailability of GLP-1.
A potentially confounding effect of this study is the method of delivery. With polypeptides that exhibit a UCST, to achieve Csats that are equivalent to the range of Csats previously tested with LCST polypeptides, the solution cloud point of the polypeptide will often be dramatically higher than the body temperature of the animal. Thus, one of our first tests will be establishing depots using either solubilizing small molecules (urea) that can diffuse from the injection site more rapidly than the polymer, thus enabling the polypeptide to be injected in a soluble solvent that rapidly exchanges with the environment to become a poor solvent, forming a depot (
Next, we designed polypeptides of similar molecular weight that exhibit different Csat. We will achieve this feat by utilizing the same parameter as before, the aromatic:aliphatic ratio to rationally tune the binodal of the GLP-1-polypeptide fusion. This allowed us to observe the effect to Csat on the therapeutic efficacy of the fusion. However, Csat is just one parameter that affects bioavailability. The overall size of the molecule is also critical to the diffusion/convection in the subcutaneous space. Thus, using similar ranges of Csat we will test depots for delivery as we increase the molecular weight of the molecule overall. Previous studies by previous lab members have demonstrated that there diminishing returns of molecular weight beyond ˜35 kDa.
Each octapeptide amino acid motif with the desired saturation concentration were genetically fused to the C terminus of GLP-1. To increase the number of total repeats of the gene, we performed iterative cloning steps of Recursive Directional Ligation by Plasmid Reconstruction adding an addition twenty repeats during each step. Transformations were performed into the desired E. coli cell line—BL21 (DE3) for recombinant expression.
Individual liquid cultures of BL21 E. coli strains each harboring our gene of interest were inoculated into 5 mL of Terrific Broth (TB) medium from frozen glycerol stocks and grown to confluence overnight (16-18 hours). Cultures were then inoculated at a 1:200 dilution in 1 L TB media supplemented with 45 μg·mL−1 kanamycin. Cells were grown at 37° C. in a shaking incubator (˜200 RPM.) for 9 hr, at which time protein expression was induced by the addition of 500 μM Isopropyl-β-D-thiogalactoside (IPTG). Cells were then incubated at 37° C. (shaking at ˜200 RPM) for an additional 18 hr. Protein was then purified from the insoluble cell suspension fraction. In brief, cell pellets were isolated by centrifuging cultures at 3500 RCF and resuspending in 20 mL of milli-Q water. Cells were then lysed by sonicating the cell solutions for 2 minutes, with 10 seconds of pulsing followed by 40 seconds of rest on ice (Misonix; Farmingdale, N.Y.)
Centrifuging each lysate suspension at 20,000 RCF for 20 minutes results in a soluble and insoluble fraction. The supernatant was discarded with the insoluble fraction resuspended in an approximately equal volume of 8 M urea+140 mM PBS (˜6-8 mL). This suspension was heated for 10 min in a 37° C. water bath and then centrifuged at 20,000 RCF for 20 minutes. The supernatant was collected from this suspension and dialyzed in a 10 kDa membrane (SnakeSkin™, Thermo Fischer Scientific) against a 1:200 milli-Q water solution at 4° C. The dialysis water was changed twice over a 48-hour period. From inside the dialysis bag, both insoluble and soluble components were collected and centrifuged at 3500 RCF for 10 minutes and 4° C. The supernatant was removed and the remaining insoluble pellet containing the protein of interest was lyophilized for a minimum of three days to remove all water from the pellet.
Protein purity was characterized by 4-20% gradient tris-HCl (Biorad, Hercules, Calif.) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and staining with either 0.5 M copper chloride or SimplyBlue™ SafeStain (Thermo Fischer Scientific). Protein yield was determined by weight after lyophilization.
Turbidity profiles were obtained for each of the constructs by recording the optical density as a function of temperature (1° C. min−1 ramp) on a temperature-controlled UV-vis spectrophotometer (Cary 300 Bio; Varian Instruments; Palo Alto, Calif.). The transition temperature (T1) was defined as the inflection point of the turbidity profile. Samples were measured in PBS at 10 μM.
All experimental procedures were conducted under protocol A053-15-02 approved by the Duke Institutional Animal Care and Use Committee (IACUC). 6-week old, male C57Bl/6J mice were purchased from Jackson Labs (strain 000664) and group housed in a room with a controlled photoperiod (12 hr light/12 hr dark cycle) and allowed at least 1 week to acclimate to the facilities prior to that start of procedures. Animals had unlimited access to water and food and were observed daily for signs and symptoms of distress. The diet-induced obese (DIO) phenotype was achieved by maintaining the mice on a high-fat (60 kcal % fat) diet upon arrival to the facility.
Constructs were endotoxin purified prior to injection by passing the solution through a sterile 0.22 μm Acrodisc filter comprised of a positively charged and hydrophilic Mustang® E membrane (Pall Corporation). Constructs were filtered in 2 M urea+140 mM PBS at 37° C. and then dialyzed against milli-Q H2O at 4° C., changing the water three separate times over the course of 72 hours. Aggregated material was removed from the dialysis bag and pelleted with centrifugation (4° C., 3500 rpm). Samples were frozen and lyophilized for a minimum of 48 hrs.
In one method, the polypeptide is resuspended at 175 μM in 2 M urea+140 mM PBS. A total volume of 200 μL is injected into the right hind flank after shaving and removing all hair with chemical dissolution at the site of injection. Mice were weighed to determine injection volume required for ˜2100 nmole of GLP-1 per kg of animal weight. Injection volume did not exceed 200 μL. In a second method, a small incision is made on the right hind flank with surgical scissors after animals were anesthetized with isoflurane. Incision site was pre-sterilized according to Duke husbandry guidelines. Pre-weighed, dehydrated polypeptide pellets are then inserted under the skin. The pellets rapidly rehydrate and become adherent to the skin tissue and thus for sealing the incision site, only a small amount of surgical glue was used to secure the skin flap.
Mice were put into a clear restraining tube. Their tails were wiped with 50% ethanol in sterile water and then dried. A small incision was made adjacent to the tail vein using a small lancet. The first drop of blood was blotted away. Blood glucose was quantified by applying the second drop of blood to the test strip of an AlphaTRAK 2 blood glucose meter (Abbott Laboratories). Weight was measured on a scale zeroed with a container into which the mice were briefly placed.
Experimental numbers for both in vitro and in vivo studies were selected based on knowledge gleaned from previous experiments or other published data. Because of the small sample size (n s 6), normality of groups was not tested. Variance across groups was similar except in untreated versus treated in vivo groups, which is not unexpected given the lack of glucose control in the mouse models tested. Blood glucose and percent change in weight studies were analyzed using repeated measures ANOVA, followed by lower order ANOVAs and Dunnett's Test for multiple comparisons. For comparing two groups, two-tailed Student's t-tests were used. No blinding was performed. Analysis and data processing were performed using Igor and R software.
We envisioned two strategies with the potential to establish depots subcutaneously for the UCST polypeptides which have transition temperatures far above safe biological temperature ranges, precluding a soluble to insoluble transition upon cooling to body temperature. The two strategies are to 1) employ urea to lower the solution cloud point for injection and 2) injection of a concentrated, dehydrated GLP-1-IDP fusion that will rehydrate, releasing peptide fusion. We decided to directly visualize this effect via fluorescent tomography. We chose a model IDP, (Gly-Arg-Gly-Asp-Ser-Pro-Tyr-Gln)-40 which has a predicted transition temperature of >70° C. at the injection concentration necessary (175 μM or 1.2 mg which corresponds to equivalent doses of GLP-1-ELP fusions used in previous studies, 1000 nmole kg−1). Using a near infrared fluorescent tag (CW800) attached via NHS-ester chemistry at free amines, we visualized the localization of the polypeptide in the hind flank.
We know that inclusion of 1 M urea in solution with the polypeptide will reduce is observed cloud by point by ˜25° C. Thus, by resuspending the model polypeptide in 2 M urea+PBS at 175 μM we can inject in a soluble state under ambient conditions. We hypothesize that considering the two order of magnitude difference in molecular weight between urea and the model polypeptide, urea will rapidly diffuse out of the sub-cutaneous space, leaving the remaining polypeptide in a poor solvent and thus will transition in situ.
Upon injection we observe something akin to a “burst” release of the polypeptide (
We also injected a dehydrated coacervate that had the same total protein content as the urea experiments. Here we observe a completely different behavior. Although our initial intention was to use convective flow from the syringe to push the dehydrated depot from the needle point into the subcutaneous space, upon contact with water, the dehydrated depot becomes extremely adhesive to the hydrophobic needle (
Fusion of GLP-1 to the N terminus of polypeptides was generally well tolerated. We observed minimal loss in yield from recombinant expression with most constructs expressing between 25-50 mg L−1. As mentioned previously, we wanted to design peptide-polypeptide fusions that have Csat in the general ranges of ˜0.1, 10, >100 μM corresponding to slow release, optimal release and near soluble release from the depot. This roughly corresponds to the Csats predicted for [3Y:V]-20, [Y:V]-20 and [3V:Y]-20. [3V:Y]-20 was not expected to exhibit phase behavior under physiologic conditions and thus six His residues were fused to the C terminus of the polypeptide and purified from the soluble fraction with chromatography.
The phase behavior of these polypeptide fusions was measured as before with temperature dependent UV-vis spectrophotometry. In determining the UCST binodal line, we identify these two proteins indeed have the desired phase behavior with GLP-1-[3Y:V]-20 exhibiting a Csat of ˜30 μM and GLP-1-[Y:V]-20 exhibiting a Csat of ˜500 μM (
After endotoxin purification, 1.2 mg of GLP-1-[3Y:V]-20, GLP-1-[Y:V]-20 and GLP-1-[3V:Y]-20-His6× were weighed and implanted in the hind flank of C57Bl/6J mice that have been fed 60% fat diet. In addition to these 3 groups, there is an additional group that received a saline injection. Over the course of the study, we measured blood glucose via tail vein blood draws at 0, 1, 2, 4, 8, 24 hrs and then each day thereafter for a total of 8 days.
The blood glucose data can be visualized in
Measurements of the body weight of the mice provide supplementary information on the efficacy of our sub-cutaneous depots (
Body weight measurements also differentiate our two depot forming fusions from one another.
The high Csat construct body weight measurements suggest that their efficacy has waned by day 5 whereas the low Csat appears to be exerting a phenotypic effect until the end of the study (day 8).
These experiments mirror similar results of optimization experiments with GLP-1-ELP depots. There were diminishing returns of polypeptides with molecular weights exceeding 35 kDa but improvements to glucose control between 20-35 kDa. Thus, we explored creating higher molecular weight variants of GLP-1-RIDP fusions.
Increasing the molecular weight of polypeptide fusions produced a series that have Csats of 0.5, 7, and 60 μM by progressively reducing the aromatic content with aliphatic substitutions. (
The blood glucose measurements of mice with 2.0 mg depots implanted in their subcutaneous space can be visualized in
The effect of molecular weight, independent of Csat, on blood glucose can be visualized in
Tracking the body weight of the mice supports the conclusions inferred from the blood glucose measurements (
In summary, we designed mimetic fusions with the GLP-1-ELP system. We also discovered multiple new routes of establishing sub-cutaneous depots with unfavorable working transition temperatures. Using these two innovations we were able to create depots that performed with similar efficacy to previous GLP-1-ELP depots, controlling blood glucose for up to 5 days in vivo in a DIO mice model.
This application claims priority to U.S. Provisional Patent Application No. 62/985,179 filed on Mar. 4, 2020, which is incorporated by reference herein in its entirety.
This invention was made with United States government support under National Institutes of Health grant number R35GM127042 and the National Science Foundation grant number DMR-17-29671. The United States government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/020591 | 3/3/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62985179 | Mar 2020 | US |
