SINGLE CELL BAR-CODING FOR ANTIBODY DISCOVERY

• US 20140357500A1
• Filed: 03/14/2014
• Published: 12/04/2014
• Est. Priority Date: 03/15/2013
• Status: Active Grant

• Current Assignee: Abvitro LLC (Bristol-Myers Squibb Company)
• Original Assignee: AbVitro Inc. (Bristol-Myers Squibb Company)
• Inventors: VIGNEAULT, Francois, WRANGHAM BRIGGS, Adrian
• Granted Patent: US 9,816,088 B2
• Time in Patent Office: Days
• US Class Current: 506/4
• 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 for heavy and IgL pairing if antibodies.

Claims

1-182. -182. (canceled)

183. A method comprising:

(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 set is complimentary to a primer of the second set;

thereby forming first and second single cell barcoded sequences.

184. The method of claim 183, wherein the first and second single cell barcoded sequences comprise a same unique barcode.

185. The method of claim 183, wherein (b) comprises extending a first anchor primer hybridized to the first cell polynucleotide, and extending a second anchor primer hybridized to the second cell polynucleotide;

wherein the first and second anchor primers are bound to the single solid support at different locations.

186. The method of claim 185, wherein the extending occurs after (a), and does not occur within a vessel of the plurality of first vessels.

187. The method of claim 185, wherein the first anchor primer and the second anchor primer do not comprise the barcode of the barcoded polynucleotide.

188. The method of claim 185, wherein the extending comprises use of a non-template terminal transferase and a template switch primer, wherein three or more identical non-template nucleotides are added to the 3′

end of the first copy and the second copy, and wherein the template switch primer comprises a universal tag, and the template switch primer is complimentary to the three or more identical non-template nucleotides added to the 3′

end of the first copy and the second copy.

189. The method of claim 183, wherein a primer of the first set comprises an overhang region, a primer of the second set comprises an overhang region, and wherein the overhang region of the primer of the second set is complimentary to the overhang region of the primer of the first set.

190. The method of claim 183, further comprising ligating an adaptor polynucleotide comprising a universal tag to the first and second copy.

191. The method of claim 183, wherein a primer of the first set comprises an overhang region comprising a universal tag;

wherein the primer of the first set comprising an overhang region comprising a universal tag is complimentary to the first copy, the second copy, or both; and

wherein a primer of the second set is complimentary to the universal tag of the primer of the first set comprising an overhang region comprising a universal tag.

192. The method of claim 183, wherein a vessel of the plurality of first vessels further comprises the barcoded polynucleotide.

193. The method of claim 192, wherein (b) further comprises copying the barcoded polynucleotide in the vessel of the plurality of first vessels, wherein the copied barcoded polynucleotide in the vessel of the plurality of first vessels comprises a region complimentary to the first copy and the second copy.

194. The method of claim 193, wherein the barcoded polynucleotide is not attached to the single solid support in a vessel of the plurality of first vessels, the plurality of second vessels, or both.

195. The method of claim 183, wherein the barcoded polynucleotide is not present in a vessel of the plurality of first vessels.

196. The method of claim 183, wherein the barcoded polynucleotide is attached to the single solid support.

197. The method of claim 183, wherein the barcoded polynucleotide is not attached to the single solid support in a vessel of the plurality of first vessels, the plurality of second vessels, or both.

198. The method of claim 183, wherein an anchor primer that is attached to the single solid support and hybridized to the barcoded polynucleotide is extended.

199. The method of claim 183, wherein the barcoded polynucleotide is annealed to an anchor primer attached to the single solid support.

200. The method of claim 183, wherein the barcoded polynucleotide is covalently attached to the single solid support.

201. The method of claim 183, further comprising lysing the single cell in a vessel of the plurality of first vessels before (b), thereby releasing the first and second cell polynucleotides from the single cell.

202. The method of claim 183, wherein the first and second cell polynucleotides are RNA

203. The method of claim 183, wherein the first and second cell polynucleotides are DNA.

204. The method of claim 183, further comprising amplifying the first and second single cell barcoded sequences.

205. The method of claim 204, further comprising sequencing the amplified first and second single cell barcoded sequences.

206. The method of claim 205, further comprising determining a first cell polynucleotide and a second cell polynucleotide to be from a same single cell when the barcode sequences of the first and second single cell barcoded sequences are the same.

207. The method of claim 183, wherein the first cell polynucleotide comprises a variable heavy chain (V_H) sequence and the second cell polynucleotide comprises a variable light chain (V_L) sequence.

208. The method of claim 183, wherein the plurality of first vessels each further comprises a protein connected to a polynucleotide encoding the protein;

(b) further comprises copying onto the solid support a third copy comprising a copy of the polynucleotide connected to the protein that encodes the protein;

(d) further comprises amplifying the third copy with the first primer set, thereby further forming a barcoded polynucleotide from the copy of the polynucleotide connected to the protein that encodes the protein, and wherein the method further comprises;

(e) sequencing

(i) one or more of the first or second single cell barcoded sequences, and

(ii) the barcoded polynucleotide from the copy of the polynucleotide connected to the protein that encodes the protein; and

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

209. 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 to interact.

210. The method of claim 209, 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 complimentary to a primer of the second primer set.

211. The method of claim 210, 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.

212. The method of claim 211, wherein the solid substrate is a bead.

213. The method of claim 209 or 210, 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.

214. The method of claim 213, wherein the first or second primer of the third primer set comprises a sample barcode.

215. The method of claim 213, wherein (e) comprises sequencing with a fourth primer set comprising a first primer complimentary to the first universal primer binding site and second primer complimentary to the second universal primer binding site.

216. The method of claim 213, 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.

217. The method of claim 209, wherein (c) further comprises producing a copy of a second cell polynucleotide from the 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.

218. The method of claim 209, wherein first cell polynucleotide from the cell comprises a light or heavy chain.

219. The method of claim 217, wherein first cell polynucleotide from the cell comprises a heavy chain and the second cell polynucleotide from the cell comprises a light chain.

220. The method of claim 209, 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.

221. The method of claim 209, wherein the plurality of proteins comprises a plurality of immune cells.

222. 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 region complementary to a same 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 complimentary to the 3 or more non-template nucleotides, thereby forming a first plurality of uniquely barcoded cDNAs;

(b) amplifying the first plurality of uniquely barcoded cDNAs, thereby forming a second plurality of uniquely barcoded cDNAs;

(c) amplifying the second 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

(d) sequencing one or more of the sequences of the library wherein the library represents an immune state of the biological sample.

223. The method of claim 222, wherein (b) comprises amplifying the first plurality of uniquely barcoded cDNAs with:

(i) a one or more first primers comprising

(A) a region that binds to a compliment 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 compliment of the first primer binding site.

224. The method of claim 223, wherein (c) comprises amplifying the second 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,

225. The method of claim 224, wherein (d) comprises sequencing with a primer set comprising a primer complimentary to the first universal primer binding site and second primer complimentary to the second universal primer binding site.

226. The method of claim 224, 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.

227. The method of claim 222, wherein the sequencing comprises high-throughput sequencing.

228. The method of claim 222, wherein the library comprises a plurality of V_H or V_L sequences representing an immune state of the biological sample.

229. The method of claim 228, further comprising determining a variance of the V_H or V_L sequences from those of a germ line.

230. The method of claim 228, 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.

231. The method of claim 230, 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.

232. The method of claim 222, further comprising producing a consensus sequence from sequences in the library comprising the same unique barcode.

233. The method of claim 232, wherein sequencing errors are minimized, eliminated, or less than 0.01%.

234. The method of claim 222, wherein amplification bias is minimized, eliminated, or less than 0.01%.

235. The method of claim 222, 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.

236. The method of claim 222, 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.

237. The method of claim 222, wherein the same region of the polynucleotides comprises a poly-A sequence.

238. The method of claim 222, wherein the region of the first plurality of primers that is complementary to a same region of the heavy or light chain polynucleotides comprises a poly-T sequence.

239. The method of claim 222, wherein the same region of the heavy or light chain polynucleotides comprises a heavy or light chain constant region sequence.

240. The method of claim 222, wherein the heavy or light chain polynucleotides comprise immunoglobulin heavy or light chain polynucleotides.

241. The method of claim 222, wherein the heavy or light chain polynucleotides comprise T-cell-receptor heavy or light chain polynucleotides.