SINGLE CELL BAR-CODING FOR ANTIBODY DISCOVERY

• US 20180127743A1
• Filed: 09/29/2017
• Published: 05/10/2018
• Est. Priority Date: 03/15/2013
• 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
• Granted Patent: US 10,392,614 B2
• Time in Patent Office: Days
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 2317/10 characterized by their source of isolation or production
• CPC Class Codes: C07K 2317/14 Specific host cells or culture conditions, e.g. components, pH or temperature
• CPC Class Codes: C07K 2317/54 F(ab')2
• CPC Class Codes: C07K 2317/55 Fab or Fab'
• CPC Class Codes: C07K 2317/56 variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12N 15/1041 Ribosome/Polysome display, e.g. SPERT, ARM
• CPC Class Codes: C12N 15/1055 Protein x Protein interaction, e.g. two hybrid selection
• CPC Class Codes: C12N 15/1062 mRNA-Display, e.g. polypeptide and encoding template are connected covalently
• 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: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• 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 2600/118 Prognosis of disease development
• CPC Class Codes: C12Q 2600/156 : Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/158 : Expression markers
• CPC Class Codes: C12Q 2600/16 : Primer sets for multiplex assays
• CPC Class Codes: G01N 33/6854 : Immunoglobulins

Abstract

Provided herein are methods and composition for immune repertoire sequencing and single cell barcoding. In some aspects, such methods may comprise steps of: (a) forming a plurality of first vessels each comprising: (i) a single cell, and (ii) a single solid support; (b) copying onto the single solid support: (i) a first copy of a first cell polynucleotide from the single cell, and (ii) a second copy of a second cell polynucleotide from the single cell; (c) forming a plurality of second vessels each comprising (i) a single solid support from the plurality of first vessels, and (ii) a barcoded polynucleotide; and (d) amplifying (i) the first copy and the second copy with a first primer set, and (ii) the barcode with a second primer set, wherein a primer of the first primer set is complementary to a primer of the second set; and (e) forming first and second single cell barcoded sequences.

Claims

1-182. -182. (canceled)

183. A method comprising:

(a) combining a plurality of cells with a plurality of proteins, each protein of the plurality connected to a polynucleotide encoding the protein,

(b) forming a plurality of first vessels each comprising;

(i) a single cell from the plurality of cells, and

(ii) a protein connected to a polynucleotide encoding the protein from the plurality of proteins;

(c) producing;

(i) a copy of a first cell polynucleotide from the single cell, and

(ii) a copy of the polynucleotide encoding the protein;

(d) adding a polynucleotide barcode to;

(i) the copy of the first cell polynucleotide to produce a barcoded copy of the first cell polynucleotide, and

(ii) the copy of the polynucleotide encoding the protein to produce a barcoded copy of the polynucleotide encoding the protein;

(e) sequencing;

(i) the barcoded copy of the first cell polynucleotide or an amplicon thereof, and

(ii) the barcoded copy of the polynucleotide encoding the protein or an amplicon thereof; and

(f) determining polypeptides encoded by sequences from (e)(i) and (e)(ii) comprising a same barcode interact.

184. The method of claim 183, wherein (d) comprises amplifying the copy of the first cell polynucleotide and the copy of the polynucleotide encoding the protein with a first primer set, and amplifying the barcode with a second primer set, wherein a primer of the first primer set is complementary to a primer of the second primer set.

185. The method of claim 184, wherein the amplifying occurs in a plurality of second vessels, each comprising a solid substrate comprising the copy of the first cell polynucleotide from the single cell and the copy of the polynucleotide encoding the protein from a vessel of the plurality of first vessels.

186. The method of claim 185, wherein the solid substrate is a bead.

187. The method of claim 183, further comprising amplifying the barcoded copy of the first cell polynucleotide and the barcoded copy of the polynucleotide encoding the protein with a third primer set before (e), wherein a first primer of the third primer set comprises a first universal primer binding site and a second primer of the third primer set comprises a second universal primer binding site.

188. The method of claim 184, further comprising amplifying the barcoded copy of the first cell polynucleotide and the barcoded copy of the polynucleotide encoding the protein with a third primer set before (e), wherein a first primer of the third primer set comprises a first universal primer binding site and a second primer of the third primer set comprises a second universal primer binding site.

189. The method of claim 188, wherein the first or second primer of the third primer set comprises a sample barcode.

190. The method of claim 188, wherein (e) comprises sequencing with a fourth primer set comprising a first primer complementary to the first universal primer binding site and second primer complementary to the second universal primer binding site.

191. The method of claim 188, wherein the third primer set amplifies the barcoded copy of the first cell polynucleotide and the barcoded copy of a polynucleotide encoding a protein from two or more single cells of the plurality of first vessels.

192. The method of claim 183, wherein (c) further comprises producing a copy of a second cell polynucleotide from the single cell in (b), wherein (d) further comprises adding a polynucleotide barcode to the copy of the second cell polynucleotide from (c), and (e)(i) further comprises sequencing the barcoded copy of the second cell polynucleotide from (d) or amplicons thereof.

193. The method of claim 183, wherein the first cell polynucleotide from the single cell comprises a light or heavy chain.

194. The method of claim 192, wherein the first cell polynucleotide from the single cell comprises a heavy chain and the second cell polynucleotide from the single cell comprises a light chain.

195. The method of claim 183, wherein the plurality of proteins is a ribosome display antigen library, a phage display library, a yeast display library, an mRNA display library, or a mammalian display library.

196. The method of claim 183, wherein the plurality of cells comprises a plurality of immune cells.

197. A method comprising:

(a) producing cDNAs from heavy or light chain polynucleotides from a plurality of immune cells from a biological sample with;

(i) a first plurality of primers each comprising a sequence complementary to a sequence downstream of the variable region of the heavy or light chain polynucleotides from the plurality of immune cells,

(ii) a reverse transcriptase comprising a non-template terminal transferase activity, wherein 3 or more identical non-template nucleotides are added to the 3′

end of the cDNAs, and

(iii) a plurality of template switch polynucleotides, each comprising;

(A) a unique barcode,

(B) a first primer binding site 5′

to the unique barcode, and

(C) a 3′

end region complementary to the 3 or more non-template nucleotides, thereby forming a plurality of uniquely barcoded cDNAs;

(b) amplifying the plurality of uniquely barcoded cDNAs, thereby forming a library of uniquely barcoded sequences comprising a variable region of the heavy (V_H) or light (V_L) chain polynucleotides; and

(c) sequencing one or more of the sequences of the library.

198. The method of claim 197, wherein (b) comprises amplifying the plurality of uniquely barcoded cDNAs with:

(i) a one or more first primers comprising

(A) a region that binds to a complement of the same region sequence of the cDNAs, and

(B) a 5′

second primer binding site; and

(ii) a second primer comprising a region that binds to the complement of the first primer binding site.

199. The method of claim 197, wherein (b) comprises further amplifying the plurality of uniquely barcoded cDNAs with:

(i) a third primer comprising a 5′

first universal primer binding site; and

(ii) a fourth primer comprising a 5′

second universal primer binding site,

200. The method of claim 198, wherein (b) comprises sequencing with a primer set comprising a primer complementary to the first universal primer binding site and second primer complementary to the second universal primer binding site.

201. The method of claim 199, wherein the third primer comprises a sample barcode sequence 3′

to the first universal primer binding site or the fourth primer comprises a sample barcode sequence 3′

to the second universal primer binding site.

202. The method of claim 197, wherein the sequencing comprises high-throughput sequencing.

203. The method of claim 197, wherein the library comprises a plurality of V_H or V_L sequences representing an immune state of the biological sample.

204. The method of claim 203, further comprising determining a variance of the V_H or V_L sequences from those of a germ line.

205. The method of claim 203, further comprising determining:

(i) a total number of unique heavy chain polynucleotides,

(ii) a total number of unique light chain polynucleotides,

(iii) the frequency of a heavy chain polynucleotide, or

(iv) the frequency of a light chain polynucleotide.

206. The method of claim 205, further comprising selecting a therapeutic antibody based on

(i) the total number of unique heavy or light chain polynucleotides,

(ii) the frequency of a heavy or light chain polynucleotide, or

(iii) a variance of a V_H or V_L sequence from that of a germ line.

207. The method of claim 197, further comprising producing a consensus sequence from sequences in the library comprising the same unique barcode.

208. The method of claim 207, wherein sequencing errors are minimized, eliminated, or less than 0.01%.

209. The method of claim 197, wherein amplification bias is minimized, eliminated, or less than 0.01%.

210. The method of claim 197, further comprising comparing the sequences of the library from the biological sample to sequences of a library from a biological sample taken from a same subject at a different time point.

211. The method of claim 197, wherein the biological sample is from a subject with a condition or disease and wherein the method further comprises comparing the sequences of the library from the biological sample to sequences of a library from a biological sample taken from a subject without the condition or disease.

212. The method of claim 197, wherein the sequence downstream of the variable region of the polynucleotides comprises a poly-A sequence.

213. The method of claim 197, wherein the region of the first plurality of primers that is complementary to sequence downstream of the variable region of the heavy or light chain polynucleotides comprises a poly-T sequence.

214. The method of claim 197, wherein the sequence downstream of the variable region of the heavy or light chain polynucleotides comprises a heavy or light chain constant region sequence.