# US 11,111,490 B2 · Systems and methods for massively parallel combinatorial analysis of single cells

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- Number: US 11,111,490 B2
- Document ID: US11111490B2
- Status: Granted
- Kind: B2
- Application number: 16/443,381
- Filed: 2019-06-17
- Published: 2019-10-17
- Granted: 2021-09-07
- 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/US11111490B2

## 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 for droplet overlap extension RT-PCR (OE-RT-PCR) of transcripts in an inducer cell expressing a human T cell receptor (TCR) and an antigen presenting cell (APC) expressing a peptide:MHC complex, the composition comprising a TCRαβ primer pool and a second probe, wherein:
the TCRαβ primer pool comprises
(i) a plurality of universal primers for an α or β constant region, wherein each of the universal primers comprises a first subsequence that is complementary to a transcript encoding an α or β constant region of the TCR and a second subsequence that has an OE-RT-PCR linker sequence, and
wherein the OE-RT-PCR linker sequence is an artificial linker sequence and is not homologous to any endogenous sequence, and wherein at least one of the universal primers comprises a sequence complementary to a transcript encoding an a constant region of the TCR and at least one of the universal primers comprises a sequence complementary to a transcript encoding a β constant region of the TCR,
wherein the plurality of universal primers comprise a primer comprising the sequence of SEQ ID NO: 20 or SEQ ID NO: 24; and
(ii) a plurality of TCR V primers comprising a first subsequence that is complementary to a transcript encoding a V segment of the TCR and a second subsequence that has the OE-RT-PCR linker sequence, and
the second probe of comprises a third subsequence that is complementary to a transcript encoding the peptide:MHC complex and a fourth subsequence that is complementary to the OE-RT-PCR linker sequence,
wherein the universal primers and the second probe are configured for OE-RT-PCR amplification of a product containing a TCR polynucleotide and a peptide:MHC polynucleotide,
wherein the plurality of TCR V primers comprise a primer comprising the sequence of SEQ ID NO: 17, 18, 19, 21, 22, or 23.

### Claim 2 (depends on 1)

The composition of claim 1, further comprising a third probe comprising a fifth subsequence that is complementary to a transcript of a T cell activation marker.

### Claim 3 (depends on 2)

The composition of claim 2, wherein the T cell activation marker is CD69 or IFNγ.

### Claim 4 (depends on 2)

The composition of claim 2, wherein the T cell activation marker is a transcriptional reporter engineered into the T cell.

### Claim 5 (depends on 2)

The composition of claim 2, wherein the third probe further comprises a sixth subsequence that is complementary to at least a part of the first probe or the second probe.

### Claim 6 (depends on 1)

The composition of claim 1, wherein the APC is HEK293 or CHO cell.

### Claim 7 (depends on 1)

The composition of claim 1, wherein the inducer cell is a primary T cell isolated from a cancer patient.

### Claim 8 (depends on 1)

The composition of claim 1, wherein the inducer cell is a cell line engineered to express the TCR.

### Claim 9 (depends on 1)

The composition of claim 1, wherein the plurality of universal primers comprise a primer having the sequence of SEQ ID NO: 20 and a primer having the sequence of SEQ ID NO: 24.

### Claim 10 (depends on 1)

The composition of claim 1, wherein the plurality of TCR V primers comprise a primer having the sequence of SEQ ID NO: 21, 22, or 23.

### Claim 11 (depends on 1)

The composition of claim 1, wherein the plurality of TCR V primers comprise: a primer having the sequence of SEQ ID NO: 21; a primer having the sequence of SEQ ID NO: 22; and a primer having the sequence of SEQ ID NO: 23.

### Claim 12 (depends on 1)

The composition of claim 1, wherein the plurality of TCR V primers comprise a primer having the sequence of SEQ ID NO: 17, 18, or 19.

### Claim 13 (depends on 1)

The composition of claim 1, wherein the plurality of TCR V primers comprise: a primer having the sequence of SEQ ID NO: 17; a primer having the sequence of SEQ ID NO: 18; and a primer having the sequence of SEQ ID NO: 19.