Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
  • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
  • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

• Proteins can be useful therapeutic agents when engineered for specificity, and selectivity for a clinical target. Their complexity, versatility, tolerability, and diversity often make them superior alternatives to small molecule drugs, and the long half-life, specificity, and selectivity can make them attractive for therapies.
• protein engineering allows for the development of potent therapeutics targeted toward a protein or receptor of interest, the body has many mechanisms with which to clear such protein therapies. Accordingly, there exists a need for reliable and broadly applicable protein delivery solutions.
• unstructured polypeptides having no discernible repeat motif wherein the polypeptide is soluble below the lower critical solution temperature (LCST), soluble above the upper critical solution temperature (UCST), or a combination thereof, wherein the LCST and UCST are each independently from about 0°C to about 100°C.
• LCST lower critical solution temperature
• UCST upper critical solution temperature
• fusion proteins comprising at least one binding polypeptide and at least one of the disclosed unstructured polypeptides.
• methods for treating a disease in a subject in need thereof comprising administering to the subject an effective amount of a disclosed fusion protein.
• methods of diagnosing a disease in a subject comprising contacting a sample from the subject with a disclosed fusion protein; and detecting binding of the fusion protein to a target to determine presence of the target in the sample, wherein the presence of the target in the sample indicates the disease in the subject.
• determining the presence of a target in a sample comprising contacting the sample with a disclosed fusion protein under conditions to allow a complex to form between the fusion protein and the target in the sample; and detecting the presence of the complex, wherein presence of the complex is indicative of the target in the sample.
• a method of determining the effectiveness of a treatment for a disease in a subject in need thereof comprising contacting a sample from the subject with a disclosed fusion protein under conditions to allow a complex to form between the fusion protein 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.
• disclosed are methods of diagnosing a disease in a subject comprising contacting a sample from the subject with a disclosed fusion protein;
• determining the level of a target in the sample 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.
• FIG. 2 is a diagram showing the amino acid compositions of exemplary non- repetitive unstructured polypeptides (SEQ ID NOs: 11-18).
• Each non-repetitive unstructured polypeptide comprises 240 amino acids.
• Each amino acid sequence comprises approximately 1/6 proline (P) residues, approximately 1/3 glycine (G) residues, and approximately 1/2 X residues, where X is one or more amino acids selected from the group consisting of valine (V), alanine (A), leucine (L), lysine (K), threonine (T), isoleucine (I), tyrosine (Y), serine (S), and phenylalanine (F).
• Each of the selected amino acids for X can occur at equal frequencies to each other (SEQ ID NOs: 11-18).
• FIG. 3(A)-(B) are graphs showing the characterization of the transition temperatures of the repetitive (SEQ ID NOs: 3-6) and exemplary non-repetitive polypeptides (SEQ ID NOs: 7- 10).
• FIG. 3(A) is a graph showing repetitive and exemplary non-repetitive polypeptides comprising 200 amino acids.
• FIG. 3(B) is a graph showing repetitive and exemplary non- repetitive polypeptides comprising 400 amino acids.
• FIG. 4(A)-(B) are graphs showing transition temperature characterization of exemplary non-repetitive unstructured polypeptides (SEQ ID NOs: 11-18).
• FIG. 4(A) is a graph showing transition temperature characterization of exemplary non-repetitive unstructured polypeptides at 25 ⁇ in PBS.
• FIG. 4(B) is a graph showing transition temperature
• Elastin-like polypeptides are repetitive polypeptides.
• ELP refers to a polypeptide comprising the pentapeptide repeat sequence (VPGXG) n , wherein X is any amino acid except proline and n is an integer greater than or equal to 1 (SEQ ID NO: 23).
• ELPs have been examined and characterized as having lower critical solution temperature (LCST) behavior.
• ELPs may include, for example, repeating subsequences of GAGVPGVGVP (SEQ ID NO: 1) or GVGVPGVGVPGAGVPGVGVPGVGVP (SEQ ID NO: 2), herein referred to as A[0.5] and A[0.2] respectively (see McDaniel, J. R. et al. (2013) Biomacromolecules, which is incorporated by reference herein in its entirety).
• A[0.2] rep-200 SEQ ID NO: 3
• (GVGVPGVGVPGAGVPGVGVPGVGVP) 8 ) includes the A[0.2] subsequence repeated 8 times for a total of 200 amino acids.
• A[0.5] rep-200 (SEQ ID NO: 4, (GAGVPGVGVP) 20 ) includes the A[0.5] subsequence repeated 20 times for a total of 200 amino acids.
• A[0.2] rep-400 (SEQ ID NO: 5, (GVGVPGVGVPGAGVPGVGVPGVGVP) ie) includes the A[0.2] subsequence repeated 16 times for a total of 400 amino acids.
• A[0.5] rep-400 (SEQ ID NO: 6, (GAGVPGVGVP) 40 ) includes the A[0.5] subsequence repeated 40 times for a total of 400 amino acids. The amino acid compositions of these sequences are depicted in FIG. 1.
• unstructured, non-repetitive polypeptides that lack the requisite pentapeptide sequence of ELPs, yet unexpectedly still have LCST behavior. Accordingly, the disclosed unstructured polypeptides lack secondary structure (according to CD) and are thermally responsive, all without having a discernable repetitive sequence within the
• the modifier "about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (for example, it includes at least the degree of error associated with the measurement of the particular quantity).
• the modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4" also discloses the range “from 2 to 4.”
• the term “about” may refer to plus or minus 10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 1 1%, and “about 1” may mean from 0.9-1.1. Other meanings of "about” may be apparent from the context, such as rounding off, so, for example "about 1” may also mean from 0.5 to 1.4.
• affinity refers to the binding strength of a binding polypeptide to its target (i.e., binding partner).
• the term "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.
• 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.
• the biomarker comprises a disease marker.
• the biomarker can be a gene that is upregulated or downregulated in a subject that has a disease.
• 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.
• the biomarker comprises a small molecule.
• the biomarker comprises a polypeptide.
• control As used herein, the terms "control,” “reference level,” and “reference” are used 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.
• AIM Adaptive Index Model
• ROC analysis 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.
• cutoff values may be determined by a quartile analysis of biological samples of a patient group.
• 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, TX; SAS Institute Inc., Cary, NC).
• the healthy or normal levels or ranges for a target or for a protein activity may be defined in accordance with standard practice.
• 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.
• the host cell includes Escherichia coli.
• reporter As used herein, the term “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.
• the reporter comprises a radiolabel.
• Reporters may include moieties that produce light, e.g., acridinium compounds, and moieties that produce fluorescence, e.g., fluorescein.
• the signal from the reporter is a fluorescent signal.
• the reporter may comprise a fluorophore.
• fluorophores examples include, but are not limited to, acrylodan (6-acryloy 1-2- 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][l,2,5]oxadiazole (NBD), Alexa fluorescent dyes, and derivatives thereof.
• acrylodan 6-acryloy 1-2
• 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” 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.
• 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.
• 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.
• the fusion protein may be used as a vaccine.
• the vaccine can be administered via electroporation, such as by a method described in U.S. Patent No. 7,664,545, the contents of which are incorporated herein by reference in its entirety.
• the electroporation can be by a method and/or apparatus described in U.S. Patent Nos. 6,302,874; 5,676,646; 6,241,701 ;
• the methods may include contacting a sample from the subject with a fusion protein as detailed herein under conditions to allow a complex to form between the fusion protein 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.
• Each of the selected amino acids for X occurs at equal frequencies to each other.
• VPGXG (VPGXG) n , wherein X is any amino acid except proline and n is an integer greater than or

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• 2017
  • 2017-09-22 JP JP2019515805A patent/JP2020500150A/ja active Pending
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