# US 11,993,865 B2 · Selection of affinity reagents

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- Number: US 11,993,865 B2
- Document ID: US11993865B2
- Status: Granted
- Kind: B2
- Application number: 17/424,435
- Filed: 2019-11-20
- Published: 2023-04-06
- Granted: 2024-05-28
- Earliest claimed: 2018-11-20
- Assignees: Nautilus Subsidiary, Inc.
- Applicants: Nautilus Subsidiary, Inc.
- Inventors: Joshua S. Klein; Jarrett Egertson; Tracy Chan; Sonal Tonapi; Parag Mallick; Minyong Chung; Abdullah Ozer; Elliott Sorelle; David Stern
- Web page: https://patentgrep.com/patent/US11993865B2

## CPC

- C40B 30/04: by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
- C40B 40/10: Libraries containing peptides or polypeptides, or derivatives thereof
- C12N 15/1048: {SELEX}
- C12N 15/1072: {Differential gene expression library synthesis, e.g. subtracted libraries, differential screening}
- G01N 33/6878: {in epitope analysis}
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Methods and systems are provided herein for selecting an affinity reagent which binds a desired peptide epitope in a plurality of sequence contexts. The method relies on obtaining a peptide library, each peptide having the sequence αXβ, wherein X is the desired peptide epitope, wherein each of α and β comprise an amino acid, using the peptide library to select an affinity reagent.

## Claims

### Claim 1

A method of identifying an affinity reagent for an epitope, comprising: obtaining a first peptide library comprising a first plurality of at least 400 unique peptides, each peptide comprising a sequence of a formula αXβ, wherein X is the desired epitope, wherein the desired epitope X is a sequence of 2 to 20 amino acids, wherein the desired epitope X is identical among the first plurality of peptides, wherein α and β are flanking domains comprising known amino acid sequences; wherein the flanking domains α and β are any sequence of 1 to 10 amino acids, and wherein the flanking domains α and β vary among the first plurality of peptides;
exposing the first peptide library to a plurality of affinity reagents, thereby binding at least one affinity reagent of the plurality of affinity reagents to the first peptide library, wherein an affinity reagent of the plurality of affinity reagents comprises oligonucleotides, peptimers, mini protein binders, antibodies, antibody fragments, or a combination thereof;
determining the sequence or sequences of the at least one affinity reagents bound to the first plurality of peptides, thereby forming a reduced affinity reagent pool;
obtaining a second peptide library comprising a second plurality of at least 400 unique peptides, each peptide comprising a sequence of a formula γXδ, wherein X is the desired epitope, wherein the desired epitope X is a sequence of 2 to 20 amino acids, wherein the desired epitope X is identical among the second plurality of peptides, wherein γ and δ are flanking domains comprising known amino acid sequences; wherein the flanking domains γ and δ are any sequence of 1 to 10 amino acids, and wherein the flanking domains γ and δ vary among the second plurality of peptides;
exposing the second peptide library to the reduced affinity reagent pool, thereby binding at least one affinity reagent of the reduced affinity reagent pool to the second peptide library; and
determining the sequence or sequences of at least one affinity reagent of the reduced affinity reagent pool bound to the second peptide library, thereby identifying an affinity reagent for the desired epitope X.

### Claim 2 (depends on 1)

The method of claim 1, wherein α and β comprise the same amino acid sequence.

### Claim 3 (depends on 1)

The method of claim 1, wherein α and β comprise differing amino acid sequences.

### Claim 4 (depends on 1)

The method of claim 1, wherein α and β comprise differing amino acid sequences from γ and δ.

### Claim 5 (depends on 1)

The method of claim 1, wherein α and β comprise amino acid sequences with homogeneous chemical diversity.

### Claim 6 (depends on 5)

The method of claim 5, wherein the amino acid sequences with homogeneous chemical diversity comprise amino acid sequences comprising two or more amino acid side groups with equivalent electrical charge, hydrophobicity, hydrophilicity, steric size, polarity, molecular structure, or a combination thereof.

### Claim 7 (depends on 1)

The method of claim 1, wherein α and β comprise amino acid sequences with heterogeneous chemical diversity.

### Claim 8 (depends on 7)

The method of claim 7, wherein the amino acid sequences with heterogeneous chemical diversity comprise amino acid sequences comprising two or more amino acid side groups with differing electrical charge, hydrophobicity, hydrophilicity, steric size, polarity, molecular structure, or a combination thereof.

### Claim 9 (depends on 1)

The method of claim 1, wherein γ and δ comprise amino acid sequences with homogeneous chemical diversity.

### Claim 10 (depends on 9)

The method of claim 9, wherein the amino acid sequences with homogeneous chemical diversity comprise amino acid sequences comprising two or more amino acid side groups with equivalent electrical charge, hydrophobicity, hydrophilicity, steric size, polarity, molecular structure, or a combination thereof.

### Claim 11 (depends on 1)

The method of claim 1, wherein γ and δ comprise amino acid sequences with heterogeneous chemical diversity.

### Claim 12 (depends on 11)

The method of claim 11, wherein the amino acid sequences with heterogeneous chemical diversity comprise amino acid sequences comprising two or more amino acid side groups with differing electrical charge, hydrophobicity, hydrophilicity, steric size, polarity, molecular structure, or a combination thereof.

### Claim 13 (depends on 1)

The method of claim 1, further comprising washing the at least one affinity reagent of the plurality of affinity reagents bound to the first peptide library.

### Claim 14 (depends on 1)

The method of claim 1, further comprising washing the at least one affinity reagent of the reduced affinity reagent pool bound to the second peptide library.

### Claim 15 (depends on 1)

The method of claim 1, wherein the peptides within the first or second peptide library are in a native state.

### Claim 16 (depends on 1)

The method of claim 1, wherein the peptides within the peptide first or second library are in a non-native state.

### Claim 17 (depends on 1)

The method of claim 1, wherein the non-native state is a denatured state or a partially-folded state.

### Claim 18 (depends on 1)

The method of claim 1, comprising repeating one or more times the steps of exposing the first peptide library to the plurality of affinity reagents and determining the sequence or sequences of the at least one affinity reagents.

### Claim 19 (depends on 1)

The method of claim 1, comprising repeating one or more times the steps of exposing the second peptide library to the reduced affinity reagent pool and determining the sequence or sequences of the at least one affinity reagents.

### Claim 20 (depends on 1)

The method of claim 1, wherein an affinity reagent of the plurality of affinity reagents comprises an oligonucleotide.

### Claim 21 (depends on 1)

The method of claim 1, wherein X is a sequence of 3 amino acids.

### Claim 22 (depends on 1)

The method of claim 1, wherein X is a sequence of 4 amino acids.

### Claim 23 (depends on 1)

The method of claim 1, wherein X is a sequence of 5 amino acids.

### Claim 24 (depends on 1)

The method of claim 1, wherein X is a sequence of 6 amino acids.

### Claim 25 (depends on 1)

The method of claim 1, wherein the affinity reagent comprises an antibody or antibody fragment.

### Claim 26 (depends on 1)

The method of claim 1, wherein the affinity reagent comprises an aptamer.