Methods and compositions for protein detection
AssigneeManifold Biotechnologies, Inc.
InventorsPierce Ogden, Gleb Kuznetsov, Shane Lofgren, Kathleen Nudel, Hoong Chuin Lim, Karen Duffy, Jeffrey Chang
Life of this patent
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Abstract
Methods and systems for quantification of an abundance of one or more payloads (e.g., proteins) in a mixture (e.g., a complex mixture (e.g., in vivo)) using barcodes (e.g., peptide barcodes), binders (e.g., polypeptide binders), and binding agents (e.g., phage) are provided herein.
Claims115
A method comprising steps of:
- a) subjecting a population of barcoded proteins to an assessment;
- b) separating those members of the population that satisfy the assessment from those that do not, so that either a positive population or a negative population, or both is identified;
- c) contacting the positive population, or the negative population, or each population separately from the other, with a set of binders which includes at least one particular binder specific for each barcode in the population;
- and
- d) determining which binders bind to the separated members, thereby determining which barcoded proteins are present in the contacted population(s).
A method comprising steps of:
- a) contacting a set of binders either with a first population, with a second population, or separately with each of the first and second populations, of barcoded proteins, wherein:
- i) each binder binds specifically (e.g., with known affinities) to one or more barcodes;
- ii) the set of binders, collectively, includes at least one binder specific for each of the barcodes in the first and second populations,
- wherein the first and second populations have been separated from one another based on performance in an assessment;
- and
- b) determining which binders of the set bind to a member of the first population, the second population, or both, thereby determining which barcoded proteins are present in the contacted population(s).
The method of any one of the preceding claims, wherein the barcode is comprised in the Complementarity-Determining Regions (CDR) of the protein.
The method of any one of the preceding claims, wherein the barcode is or comprises an amino acid, and wherein the barcode is attached to the protein.
The method of any one of the preceding claims, wherein a binder is or comprises a binding moiety displayed on a phage.
The method of any one of the preceding claims, wherein the barcode is synthetic.
The method of any one of the preceding claims, wherein the barcode is 1-100, 5-50, 8-25, 9-25, or 9-15 amino acids in length.
The method of any one of the preceding claims, wherein the barcode is 10 amino acids in length.
The method of any one of the preceding claims, wherein the barcode has relatively no effect on protein function.
The method of any one of the preceding claims, wherein the barcode does not illicit an immune response.
The method of any one of the preceding claims, wherein the barcodes are orthogonal to each other.
The method of any one of the preceding claims, wherein the barcode is attached to a suitable position on the protein.
The method of any one of the preceding claims, comprising separating the binders that bind to at least one protein.
The method of any one of the preceding claims, wherein the step of determining comprises performing one or more of amplification, propagation, and sequencing (e.g., nucleic acid (e.g., DNA, RNA) amplification, propagation, and/or sequencing).
The method of any one of the preceding claims, wherein the step of determining comprises quantifying the number of binders that bind to a barcoded protein, wherein the quantifying is performed by decoding the nucleotide sequence of each binder that binds to the barcoded protein.
The method of any one of the preceding claims, wherein the barcoded proteins are in a complex mixture.
The method of any one of the preceding claims, wherein the barcoded proteins are in a purified sample.
A nucleic acid whose nucleotide sequence is or comprises a sequence encoding a peptide barcode characterized in that:
- a) the peptide barcode has a length within a range of 1 to 100, 5 to 50, 8 to 25, 9 to 25, or 9 to 15 amino acids;
- and
- b) has been determined to bind specifically to a particular group of polypeptide binders within a set of binders.
A nucleic acid whose nucleotide sequence is or comprises a sequence encoding a polypeptide binder moiety characterized in that:
- a) the polypeptide binder moiety has a length within a range of 10 to 400 amino acids;
- and
- b) has been determined to bind specifically to a particular group of peptide barcodes within a collection of barcodes.
A set of barcode and binders, wherein:
- a) each barcode is a peptide between 1 to 100, 5 to 50, 8 to 25, 9 to 25, or 9 to 15 amino acids in length that binds specifically to a particular group of binders among the binders in the set;
- and
- b) each binder is a polypeptide that binds specifically to at least one barcode among the barcodes in the set.
A kit comprising:
- a) a set of binders, each of which is a polypeptide that binds specifically to at least a particular peptide barcode in a collection barcodes, wherein each binder is provided as a polypeptide, a nucleic acid encoding the polypeptide, or both.
A method of pharmacokinetic screening, the method comprising:
- a) injecting a set of barcoded therapeutic candidate proteins into an animal, wherein each therapeutic candidate protein comprises a specific peptide barcode;
- b) obtaining a sample from the animal;
- c) purifying one or more barcoded therapeutic candidate proteins from the sample;
- d) contacting the sample with a set of binders (e.g., binding agents with binders expressed on them) which includes at least one particular binder specific for each barcode in the sample;
- and
- e) determining (e.g., simultaneously) the relative amounts of each binder present in the sample to determine each barcoded therapeutic candidate proteins' pharmacokinetic properties, biodistribution, half-life, tissue-mediated drug disposition (TMDD), or in vivo stability.
The method of any one of 75 to 104, wherein the animal is genetically modified to express the barcoded proteins.
A method of characterizing a collection of peptide barcodes, the method comprising:
- a) providing:
- (i) a library of phage particles, wherein each phage particle is designed to express a polypeptide binder, and wherein each binder binds to one or more peptide barcodes;
- (ii) a collection of peptide barcodes;
- b) contacting each phage particle with each barcode to form bound phage-barcode particles;
- c) determining the amount of binding between each phage particle and barcode;
- and
- d) identifying phage-barcode pairs that bind specifically to each other among the barcodes in the collection and the phages in the library.
A method of characterizing a collection of peptide barcodes, the method comprising:
- a) providing:
- (i) a set of binders, wherein each binder is a polypeptide that binds to one or more peptide barcodes;
- (ii) a collection of peptide barcodes;
- b) contacting each binder with each barcode to form bound binder-barcode particles;
- c) determining the relative amount of binding between each polypeptide binder and peptide barcode;
- and
- d) identifying binder-barcode pairs that bind specifically to each other among the barcodes in the collection and the binders in the set.
A database of amino acid or encoding nucleic acid sequences for a collection of peptide barcodes, which database is embodied in a computer readable format, wherein each barcode sequence:
- a) has a length within a range of 1 to 100, 5 to 50, 8 to 25, 9 to 25, or 9 to 15 amino acids;
- b) has been determined to bind specifically to one or more polypeptide binders within a set of binders that each bind specifically to one or more of the barcodes in the collection.
A database of amino acid or encoding nucleic acid sequences for a set of polypeptide binders, which database is embodied in a computer readable format, wherein each binder sequence:
- a) has a length within a range of 10 to 400 amino acids;
- b) has been determined to bind specifically to one or more peptide barcodes within a collection of barcodes that each bind specifically to one or more of the binders in the set.
A database of amino acid or encoding nucleic acid sequences for a set of barcode-binder associations, embodied in a computer readable format, wherein:
- a) each barcode is a peptide between 1 to 100, 5 to 50, 8 to 25, 9 to 25, or 9 to 15 amino acids in length;
- and
- b) each binder is a polypeptide that binds specifically to one or more barcodes among the barcodes in the set.
A set of barcode-binder association designations, embodied in a computer readable format, wherein:
- a) each barcode is a peptide between 1 to 100, 5 to 50, 8 to 25, 9 to 25, or 9 to 15 amino acids in length;
- and
- b) each binder is a polypeptide that binds specifically to one or more barcodes among the barcodes in the set.
A method of treatment comprising:
- administering a therapeutic protein that has been determined to satisfy an assessment by a process comprising steps of:
- a) subjecting a population of barcoded proteins to the assessment;
- b) separating those members of the population that satisfy the assessment from those that do not, so that either a positive population or a negative population, or both is identified;
- c) contacting the positive population, or the negative population, or each population separately from the other, with a set of binders which includes at least one particular binder specific for each barcode in the population;
- d) determining which binders bind to the separated members, thereby determining which barcoded proteins are present in the contacted population(s);
- and
- e) identifying the therapeutic protein from the barcoded proteins determined to be present in the contacted population(s).
A method of treatment comprising:
- administering a therapeutic protein that has been determined to satisfy an assessment by a process comprising steps of:
- a) contacting a set of binders either with a first population, with a second population, or separately with each of the first and second populations, of barcoded proteins, wherein:
- i) each binder binds specifically to one barcode relative to the other barcodes;
- and
- ii) the set of binders, collectively, includes a binder specific for each of the barcodes in the first and second populations,
- wherein the first and second populations have been separated from one another based on performance in the assessment;
- b) determining which binders of the set bind to a member of the first population, the second population, or both, thereby determining which barcoded proteins are present in the contacted population(s);
- and
- c) identifying the therapeutic protein from the barcoded proteins determined to be present in the contacted population(s).
Dependencies are inferred from the claim text.