# US 11,078,478 B2 · Methods of utilizing recombination for the identification of binding moieties

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- Number: US 11,078,478 B2
- Document ID: US11078478B2
- Status: Granted
- Kind: B2
- Application number: 16/298,374
- Filed: 2019-03-11
- Published: 2020-01-30
- Granted: 2021-08-03
- Earliest claimed: 2013-12-04
- Assignees: AxioMx, Inc.
- Applicants: AxioMx, Inc.
- Inventors: Michael Weiner; Margaret Kiss
- Web page: https://patentgrep.com/patent/US11078478B2

## CPC

- C12N 15/1037: {Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display}
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C40B 30/06: by measuring effects on living organisms, tissues or cells
- C07K 2317/622: Single chain antibody (scFv)
- C40B 30/04: by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- 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)

## Abstract

The identification of binding moieties capable of selectively interacting with one or more target antigens is of scientific, medical, and commercial value. Disclosed herein are methods and compositions for the identification, labeling and/or retrieval of cognate binding moieties.

## Claims

### Claim 1

A method of identifying a cognate binding moiety of one or more antigens, said method comprising the steps of:
a) providing
1) a population of cells, each of said cells comprising:
i) a cell surface antigen,
ii) a nucleic acid comprising a first recombination motif, and
iii) a conditionally expressed infection apparatus; and
2) a plurality of virions, each of said virions comprising:
i) one of a plurality of transgenic viral surface binding moieties and
ii) a nucleic acid comprising a second recombination motif capable of integrating with said first motif;
b) contacting said population of cells with said virions;
c) incubating said cells with said virions under conditions that are not permissive for infection but are sufficient to allow binding of one or more of said cell surface antigens by one or more of said cognate binding moieties, whereby one or more of said cells are bound by one or more of said virions;
d) emulsifying said bound cells, thereby encapsulating one or more of said cells in one or more emulsion droplets;
e) incubating said emulsified cells under conditions permissive to expression of said conditionally expressed infection apparatus, thereby allowing infection of one or more of said emulsified cells by one or more virions present in the same emulsion droplet;
f) incubating said cells in a manner sufficient to allow recombination between said first and second site-specific recombination motifs in infected cells, thereby generating a recombinant product; and
g) identifying a cognate binding moiety of one or more of said antigens by identifying the nucleic acid sequence of said recombinant product or an identifying fragment thereof, in a cell, and identifying from said nucleic acid sequence an antigen and a cognate binding moiety.

### Claim 2 (depends on 1)

The method of claim 1, wherein said cell comprises a recombination enzyme.

### Claim 3 (depends on 1)

The method of claim 1, wherein the nucleic acid comprised by one or more of said virions encodes a recombination enzyme and said incubation is sufficient to allow expression of said encoded recombination enzyme.

### Claim 4 (depends on 2)

The method of claim 2, wherein said recombination enzyme is an integrase.

### Claim 5 (depends on 1)

The method of claim 1, wherein: (i) one or more of said recombination motifs is a site-specific recombination motif; (ii) each of said cells comprises one of a plurality of distinct cell surface antigens; and/or (iii) one or more of said cell surface antigens are transgenic.

### Claim 6 (depends on 1)

The method of claim 1, wherein
each of said cells comprises one of a plurality of cell surface antigens;
the nucleic acid of a) 1) ii) comprises a first marker gene fragment adjacent to the first recombination motif, wherein the first recombination motif is a site-specific recombination motif;
each of said cells comprises a recombination enzyme; and
the nucleic acid of a) 2) ii) comprises a second marker gene fragment adjacent to the second recombination motif capable of integrating with said first motif, wherein the second recombination motif is a site-specific recombination motif;
wherein said first marker gene fragment and said second marker gene fragment are positioned such that site-specific recombination between said first and second motif will result in a functional marker gene;
wherein the recombinant product generated in step (f) is capable of expressing a functional marker protein;
and wherein after step (f) and prior to step (g) the method further comprises a step of incubating said population of cells in a manner sufficient to allow detection of cells having a phenotype resulting from expression of said functional marker protein and isolating one or more cells having said detectable phenotype.

### Claim 7 (depends on 6)

The method of claim 6, wherein one or more of said first marker gene fragments or one or more of said second marker gene fragments comprise a functional marker gene promoter and one or more of said first marker gene fragments or one or more of said second marker gene fragments comprise a coding segment of the same functional marker gene.

### Claim 8 (depends on 7)

The method of claim 7, wherein (i) one of said first marker gene fragment or said second marker gene fragment comprises a promoter gene fragment and the other comprises a coding gene fragment or (ii) one of said first marker gene fragment or said second marker gene fragment comprises a promoter gene fragment and a 5′ coding gene fragment and the other comprises a 3′ coding gene fragment.

### Claim 9 (depends on 6)

The method of claim 6, wherein said marker gene is an antibiotic resistance gene or wherein said marker gene encodes a fluorescent protein.

### Claim 10 (depends on 9)

The method of claim 9, wherein said antibiotic resistance gene is an ampicillin resistance gene.

### Claim 11 (depends on 6)

The method of claim 6, wherein (i) the polynucleotide sequence of one or more of said cell surface antigens is adjacent to a first marker gene fragment; and/or (ii) the polynucleotide sequence of one or more of said transgenic viral surface binding moieties is adjacent to a second marker gene fragment.

### Claim 12 (depends on 6)

The method of claim 6, wherein one or more of said cell surface antigens are comprised by a cell surface antigen fusion protein.

### Claim 13 (depends on 1)

The method of claim 1, wherein one or more of said transgenic viral surface binding moieties are viral surface binding moiety fusion proteins.

### Claim 14 (depends on 1)

The method of claim 1, wherein said cells are bacterial, yeast, or mammalian cells.

### Claim 15 (depends on 1)

The method of claim 1, wherein said virions are attachment-defective bacteriophage, baculovirus, or adenovirus virions.

### Claim 16 (depends on 1)

The method of claim 1, wherein said cells are E. coli cells, said infection apparatus is an F pilus apparatus, and said virions are M13 bacteriophage.

### Claim 17 (depends on 1)

The method of claim 1, wherein said virions are attachment-defective.

### Claim 18 (depends on 1)

The method of claim 1, wherein said emulsion further comprises a cleavage reagent capable of facilitating infection under permissive conditions.

### Claim 19 (depends on 1)

The method of claim 1, further comprising breaking said emulsion subsequent to said infection of one or more of said emulsified cells.

### Claim 20 (depends on 1)

The method of claim 1, wherein said antigen comprises one or more modified amino acids.

### Claim 21 (depends on 20)

The method of claim 20, wherein one or more of said modified amino acids are phosphoserines.

### Claim 22 (depends on 1)

The method of claim 1, wherein one or more of said binding moieties are antibodies.

### Claim 23 (depends on 1)

The method of claim 1, wherein (i) one or more of said binding moieties are scFv binding moieties; (ii) one or more of said viral surface binding moieties comprise one or more segments derived from the sequence of an antibody expressed by a cell of an inoculated subject, (iii) one or more of said viral surface binding moieties comprise one or more segments derived from the sequence of an antibody expressed by a naïve cell; (iv) one or more of said antigens is an antigen isolated from a pathogen; (v) said recombinant product comprises one or more variable priming sequences; (vi) said recombinant product comprises two variable priming sequences that flank the sequences of said antigen and said binding moiety; and/or (vii) said sequencing is deep sequencing or NextGeneration sequencing.

### Claim 24 (depends on 1)

The method of claim 1, wherein a segment of one or more polynucleotides encoding an antigen or binding moiety is distinguished from one or more substantially identical segments by substitution at one or more nucleobases, wherein said substitution does not modify the polypeptide encoded by the segment.

### Claim 25 (depends on 24)

The method of claim 24, wherein said one or more substituted nucleobases is a nucleobase present in a codon.

### Claim 26 (depends on 1)

The method of claim 1, wherein the conditionally expressed infection apparatus is a temperature sensitive F pilus construct.

### Claim 27 (depends on 26)

The method of claim 26, wherein the condition that is not permissive for infection is a temperature regimen.

### Claim 28 (depends on 27)

The method of claim 27, wherein the temperature regimen that is not permissive is incubation at 16° C.

### Claim 29 (depends on 26)

The method of claim 26, wherein the condition permissive to expression of the conditionally expressed infection apparatus is a temperature regimen.

### Claim 30 (depends on 29)

The method of claim 29, wherein the temperature regimen permissive to expression is incubation at 37° C.

### Claim 31 (depends on 6)

The method of claim 6, wherein said antigen comprises one or more modified amino acids.

### Claim 32 (depends on 31)

The method of claim 31, wherein one or more of said modified amino acids are phosphoserines.

### Claim 33 (depends on 6)

The method of claim 6, wherein one or more of said binding moieties are antibodies.

### Claim 34 (depends on 6)

The method of claim 6, wherein (i) one or more of said binding moieties are scFv binding moieties; (ii) one or more of said viral surface binding moieties comprise one or more segments derived from the sequence of an antibody expressed by a cell of an inoculated subject, (iii) one or more of said viral surface binding moieties comprise one or more segments derived from the sequence of an antibody expressed by a naïve cell; (iv) one or more of said antigens is an antigen isolated from a pathogen; (v) said recombinant product comprises one or more variable priming sequences; (vi) said recombinant product comprises two variable priming sequences that flank the sequences of said antigen and said binding moiety; and/or (vii) said sequencing is deep sequencing or NextGeneration sequencing.

### Claim 35 (depends on 6)

The method of claim 6, wherein the conditionally expressed infection apparatus is a temperature sensitive F pilus construct.

### Claim 36 (depends on 35)

The method of claim 35, wherein the condition that is not permissive for infection is a temperature regimen.

### Claim 37 (depends on 36)

The method of claim 36, wherein the temperature regimen that is not permissive is incubation at 16° C.

### Claim 38 (depends on 35)

The method of claim 35, wherein the condition permissive to expression of the conditionally expressed infection apparatus is a temperature regimen.

### Claim 39 (depends on 38)

The method of claim 38, wherein the temperature regimen permissive to expression is incubation at 37° C.