AFFINITY-OLIGONUCLEOTIDE CONJUGATES AND USES THEREOF

• US 20190025304A1
• Filed: 09/24/2016
• Published: 01/24/2019
• Est. Priority Date: 09/24/2015
• Status: Active Grant

• Current Assignee: Abvitro LLC (Bristol-Myers Squibb Company)
• Original Assignee: Abvitro LLC (Bristol-Myers Squibb Company)
• Inventors: Vigneault, Francois, Briggs, Adrian Wrangham, Goldfless, Stephen J., Belmont, Brian J.
• Granted Patent: US 11,156,611 B2
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: G01N 2333/7051 T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: G01N 2333/70514 CD4
• CPC Class Codes: G01N 2333/70517 CD8
• CPC Class Codes: G01N 2333/70539 MHC-molecules, e.g. HLA-molecules
• CPC Class Codes: G01N 2458/10 Oligonucleotides as tagging agents for labelling antibodies
• CPC Class Codes: G01N 33/56972 White blood cells
• CPC Class Codes: G01N 33/6872 Intracellular protein regulatory factors and their receptors, e.g. including ion channels

Abstract

Provided herein are methods and composition for single cell characterization using affinity-oligonucleotide conjugates.

Claims

1. A method for characterizing cells comprising:

performing a reaction in at least one single vessel of a plurality of vessels, the reaction comprising attaching a first vessel barcoded polynucleotide, which comprises a first vessel barcode sequence, to an oligonucleotide portion of an affinity-oligonucleotide conjugate, wherein an affinity portion of the affinity-oligonucleotide conjugate binds to a target antigen expressed by a single cell isolated in the at least one single vessel of the plurality of vessels, and wherein the oligonucleotide portion comprises an antigen identification sequence (AID).

6. The method of claim 1, wherein the single cell is from a plurality of cells from a single sample.

9. The method of claim 1, wherein the AID is barcoded to the target antigen or the affinity portion of the affinity-oligonucleotide conjugate.

18. The method of claim 1, wherein the method further comprises contacting a plurality of cells comprising the single cell with the affinity-oligonucleotide conjugate before the single cell is isolated in the at least one single vessel.

29. The method of claim 1, wherein the first vessel barcode sequence of the first vessel barcoded polynucleotide or an amplicon thereof in any one single vessel of the plurality of vessels is unique to the one single vessel.

30. The method of claim 1, wherein the method further comprises lysing the single cell in the at least one single vessel and attaching a second vessel barcoded polynucleotide comprising a second vessel barcode sequence to a cell polynucleotide from the single cell.

31. The method of claim 30, wherein attaching the first vessel barcoded polynucleotide to the oligonucleotide portion of the affinity-oligonucleotide conjugate and attaching the second vessel barcoded polynucleotide to the cell polynucleotide are performed simultaneously.

41. The method of claim 30, wherein the oligonucleotide portion of the first or the second affinity-oligonucleotide conjugate further comprises at least one of an affinity molecular barcode (AMB) sequence, a primer binding sequence, a fusion sequence, or a constant sequence.

46. The method of claim 30, wherein the method further comprises sequencing the oligonucleotide portion of the affinity-oligonucleotide along with the attached vessel barcoded polynucleotide, complements thereof, amplified products thereof, or a combination thereof, thereby producing oligonucleotide sequence reads and/or sequencing the cell polynucleotide along with the attached vessel barcoded polynucleotide, complements thereof, amplified products thereof, or a combination thereof, thereby producing cell polynucleotide sequence reads.

56. The method claim 46, wherein the method further comprises comparing oligonucleotide sequence reads to the cell polynucleotide sequence reads, vessel barcode sequences of oligonucleotide sequence reads to vessel barcode sequences of the cell polynucleotide sequence reads, antigen identification (AID) sequences of oligonucleotide sequence reads to affinity molecular barcode (AMB) sequences of oligonucleotide sequence reads, or cell polynucleotide sequence reads;

and/or analyzing vessel barcode sequences of the cell polynucleotide sequence reads or molecular barcode sequences of the cell polynucleotide sequence reads.

61. The method of claim 56, wherein the method further comprises determining a characteristic of a cell based on the analyzing or the comparing.

64. The method of claim 30, wherein the first vessel barcode sequence of the first vessel barcoded polynucleotide attached to the oligonucleotide portion of the affinity-oligonucleotide conjugate and the second vessel barcode sequence of the second vessel barcoded polynucleotide attached to the cell polynucleotide are the same and/or are from a same template vessel barcoded polynucleotide in the at least one single vessel.

70. The method of claim 1, wherein the at least one single vessel is a well, an emulsion, or a droplet.

73. The method of claim 30, wherein the method further comprises attaching a molecular barcoded polynucleotide comprising a molecular barcode sequence to the cell polynucleotide, wherein the molecular barcode sequence is barcoded to the cell polynucleotide and/or amplicons thereof.

75. The method of claim 30, wherein attaching the first vessel barcoded polynucleotide to the oligonucleotide portion or attaching the second vessel barcoded polynucleotide to the cell polynucleotide comprises a ligation reaction, an enzymatic reaction, a hybridization reaction, an extension reaction, or an amplification reaction.

79. The method of claim 1, wherein the affinity portion of the affinity-oligonucleotide conjugate binds to an extracellular antigen of the single cell.

81. The method of claim 79, wherein the extracellular antigen of the single cell is an antigen specific to or expressed by a T-cell or a B cell.

84. The method of claim 81, wherein the extracellular antigen is selected from the group consisting of CD154, CD4, CD8, CD137, CD40L, CD80, CD86, CDIIc, CD25, CD69, CD44, CD125, CD2, CD3, CDS, CD14 and CD19.

98. The method of claim 1, wherein the affinity portion of the affinity oligonucleotide conjugate is an antibody or fragment thereof, a peptide, a protein, an aptamer, a small molecule, a drug, or a cell.

106. The method of claim 98, wherein the affinity portion of the affinity oligonucleotide conjugate comprises one of a major histocompatibility complex (MHC) or functional or binding portion thereof, or a peptide that binds to an antibody or a chimeric antigen receptor (CAR).

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114. A composition comprising a plurality of vessels, wherein at least one single vessel of the plurality of vessels comprises

a. a single cell from a sample comprising a plurality of cells, and

b. a vessel barcoded polynucleotide or complement thereof comprising a vessel barcode sequence;

and

c. an affinity-oligonucleotide conjugate that binds to a target antigen of the single cell.

120. The composition of claim 114, wherein the vessel barcoded polynucleotide or a complement thereof is attached to the oligonucleotide portion of the affinity-oligonucleotide conjugate.

121. The composition of claim 114, wherein the single cell is lysed.

115-119. -119. (canceled)

122. (canceled)

123. A kit comprising:

(a) a first container comprising a first oligonucleotide comprising a first antigen identification (AID) sequence, wherein the first AID sequence is a known sequence;

(b) a second container comprising a second oligonucleotide comprising a second antigen identification (AID) sequence, wherein the second AID sequence is a known sequence and is different than the first AID sequence;

(c) one or more third containers comprising reagents capable of conjugating the first oligonucleotide to a first affinity molecule and reagents capable of conjugating the second oligonucleotide to a second affinity molecule;

(d) a set of instructions describing how to conjugate the first oligonucleotide to the first affinity molecule and the second oligonucleotide to the second affinity molecule.

124. A kit comprising:

(a) a first container comprising an oligonucleotide comprising an antigen identification (AID) sequence, wherein the AID sequence is a known sequence;

(b) a second container comprising reagents capable of conjugating the oligonucleotide to an affinity molecule;

(c) a third container comprising a plurality of vessel barcoded polynucleotides; and

(d) a set of instructions describing how to attach a vessel barcoded polynucleotide of the plurality of vessel barcoded polynucleotides to the oligonucleotide when conjugated to the affinity molecule.