# US 11,441,240 B2 · Systems and methods for massively parallel combinatorial analysis of single cells

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- Number: US 11,441,240 B2
- Document ID: US11441240B2
- Status: Granted
- Kind: B2
- Application number: 16/414,548
- Filed: 2019-05-16
- Published: 2019-12-26
- Granted: 2022-09-13
- Earliest claimed: 2017-03-13
- Assignees: GigaGen, Inc.
- Applicants: GigaGen, Inc.
- Inventors: David Scott Johnson; Adam Shultz Adler; Matthew James Spindler; Rena Aviva Mizrahi
- Web page: https://patentgrep.com/patent/US11441240B2

## CPC

- C40B 40/08: Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 14/70539: {MHC-molecules, e.g. HLA-molecules}
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 16/005: {constructed by phage libraries}
- C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags}
- C12N 15/1075: {by coupling phenotype to genotype, not provided for in other groups of this subclass}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6869: Methods for sequencing
- C12Q 1/6888: for detection or identification of organisms
- C40B 50/06: Biochemical methods, e.g. using enzymes or whole viable microorganisms
- 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)
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

Provided herein are methods that enable parallel evaluation of multiple functional nucleic acids in individual cells or subpopulations of cells, in the context of incubation with other types of single cells. The key insight is concurrent measurement of polynucleic acids derived from small populations of at least two different cell types, such that function in one cell type is linked to the clonal identity of another cell. These methods simultaneously process thousands, millions, or more single cells or small populations of cells. The method integrates molecular, algorithmic, and engineering approaches. This invention has broad and useful application in a number of biological and medical fields, including immunology and drug discovery.

## Claims

### Claim 1

A composition comprising a library of hybridized polynucleic acids that comprise a transcript of an antibody immunoglobulin heavy chain covalently linked to an activation transcript indicative of B cell activation,
wherein the hybridized polynucleic acids comprise a plurality of transcripts isolated and captured from B cells from a human subject exposed to an antigen,
wherein the B cells were isolated into an aqueous-in-oil microdisperse emulsion microdroplets, wherein each microdroplet comprises a single B cell,
wherein the activation transcript is an endogenous transcript of the B cells or a transcript of a reporter engineered into the B cells, and
wherein the hybridized polynucleic acids are indicative of transcriptional change in the B cells after incubation with the one or more inducer cells.

### Claim 2 (depends on 1)

The composition of claim 1, comprising hybridized polynucleic acids of at least 10,000 unique sequences.

### Claim 3 (depends on 2)

The composition of claim 2, comprising hybridized polynucleic acids of at least 1,000,000 unique sequences.

### Claim 4 (depends on 1)

The composition of claim 1, wherein the activation transcript is a transcript of Ki-67.

### Claim 5 (depends on 1)

The composition of claim 1, wherein the B cells are memory B cells.