Method for recovering two or more genes, or gene products, encoding an immunoreceptor

Abstract

The present invention is related to a method for recovering two or more genes, or gene products, or cDNAs, encoding for an immunoreceptor having two or more subunits, which two or more genes, or gene products, are comprised in a given source cell. The invention is further related to a method of creating a library of expressor cells, in which library each cell is capable of expressing two or more genes, or gene products, encoding for the subunits of the immunoreceptor. The invention is further related to a method of screening a library of expressor cells as created according to the above method, for one cell that expresses an immunoreceptor that has specificity for a given target molecule.

Claims

1. A method for generating a library of expressor cells where each expressor cell is capable of expressing two or more genes or gene products encoding for the subunits of an immunoreceptor, said method comprising: 1) preparing a single cell for RT-PCR by a) encapsulating an isolated human memory B-cell comprising an Ig-light chain and an Ig-heavy chain pair with an mRNA capture bead and lysis buffer in a first droplet; b) lysing the B-cell thereby releasing cellular mRNA into the first droplet; c) capturing the cellular mRNA on the mRNA capture bead such that the released cellular mRNA is bound to the mRNA capture bead inside the first droplet; d) isolating the captured cellular mRNA bound to the mRNA capture bead from the first droplet and reverse transcribing the captured cellular mRNA on the mRNA capture bead thereby forming a cDNA-loaded capture bead, wherein the reverse transcribing occurs in solution; e) encapsulating the cDNA-loaded capture bead in a second droplet; f) amplifying the cDNA of the cDNA-loaded capture bead in the second droplet and forming an amplification product, wherein the amplification product comprises an Ig-light chain sequence and an Ig-heavy chain sequence; g) linking the Ig-light chain and Ig-heavy chain sequences; and h) cloning the linked Ig-light chain and Ig-heavy chain sequences into a vector; 2) generating a library of expressor cells using the vector generated in h), wherein the expressor cells comprise the vector by transducing the vector into an expressor cell, encapsulating the expressor cell in a droplet, wherein the expressor cell expresses an immunoreceptor encoded by the Ig-light and Ig-heavy chain sequences; 3) screening the library of expressor cells for a cell that expresses an immunoreceptor by: a) culturing the library of expressor cells with a target molecule and detecting binding of the target molecule to the expressor cells; or b) detecting an antibody produced by one or more of the expressor cells from the library, thereby identifying an expressor cell that expresses an immunoreceptor, wherein said method is capable of obtaining the immunoreceptor pairs from a collection of source cells having more than 100,000 distinct specificities with high efficiency.

2. The method of claim 1, further comprising, after step d), the steps of i) monitoring cDNA synthesis by reverse transcription on the mRNA capture bead, and ii) excluding aggregated mRNA capture beads from non-aggregated mRNA capture beads.

3. The method of claim 1, wherein the linking of the Ig-light chain sequence and Ig-heavy chain sequence in step g) is accomplished by overlap extension PCR.

4. The method of claim 1, wherein the amplification product is an antibody.

5. The method of claim 1, wherein the vector is a 2A peptide-linked multicistronic vector with or without combination with an internal ribosomal entry site sequence.

6. The method of claim 1, wherein the first droplet comprises one human memory B-cell.

7. The method of claim 1, wherein the first droplet or second droplet comprises a material selected from the group consisting of: a) hydrogel-forming polymers, wherein the hydrogel-forming polymers are poly(diallyldimethylammonium chloride), poly(ethyleneimine), polylysine, polyacrylamides or acrylic acids; b) cellulose derivatives, wherein the cellulose derivatives are carboxymethylcellulose, cellulose esters; and c) polysaccharides, wherein the polysaccharides are agaroses, alginates, carrageenans, pectinates, or chitosans.

8. The method of claim 1, wherein the first droplet or second droplet is an aqueous droplet in a water/oil emulsion.

9. The method of claim 1, wherein the human memory B-cell is from a collection of mature B cells, wherein the collection of mature B cells comprises cells from the same or different donors.

10. The method of claim 1, wherein the immunoreceptor is an antibody.

11. The method of claim 1, wherein the method is a high-throughput method.

12. The method of claim 1, wherein at least 20,000 single cells are prepared for the RT-PCR in step 1.

13. The method of claim 1, wherein the expressor cells in step 2) express a copy of the Ig-light chain and the Ig-heavy chain pair of the human memory B-cell in step 1)a).

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: Microphotograph of Co-Encapsulation in Microreactors

(2) Microreactors (also called “NanoLiter Reactors” or “NLR” herein, although these reactors can also have picoliter volumes) of a single cell (C, surrogate B-cell expressing antibody and marker gene GFP for better visibility is shown here) with mRNA capture matrix Dynabeads (DB). Surrogate B cells were generated by stable transduction of CHO-K1 cells with lentivectors bearing an attenuated bi-cistronic human IgG expression cassette leading to low IgG expression levels to match Ig-expression in original human memory B cells. Attenuation was achieved by positioning of the bi-cistronic antibody expression cassette behind the stop codon of the CMV-promoter-EGFP cassette. Surrogate B cells, as used herein faithfully reflect the behavior of B cells, or T-cells.

(3) FIG. 2: Schematic Overview on Immunoglobulin V-Region PCR Strategy

(4) 1.sup.st PCR: Overlap extension PCR (oePCR) for amplification and linkage of immunoglobulin variable heavy and light chains in a “tail to head” orientation of V-heavy and V-light regions. Linker primers for OE PCR were of the following configuration.

(5) Ig-variable heavy chain: linker primer 3′ Sall J H: tattcgcactgcgcggcGTCGACgc-(J H family specific sequences)

(6) Ig-variable kappa light chain: linker primer 5′ XbaI Vk: gccgcgcagtgcgaataTCTAGAtgt-(FW1-V-kappa family specific sequences), linker primer 5′ XbaI Vi: gccgcgcagtgcgaataTCTAGAtgt-(FW1-V-lambda family specific sequences).

(7) Additional primers used were 5′L(SP)-VH(1-6) primers, and 3′Cκ 543 and 3′Cλ. Capital letters indicate the restriction sites used for insertion of the polynucleotide cassette providing the constant part of IgH, a cleavage site for later proteolytic cleavage and a signal peptide for secretion of IgKappa/lambda in a later cloning step (see below). Small letters indicate the linker sequences used in overlap extension PCR to link heavy- and light chain constructs.

(8) 2.sup.nd PCR: Nested PCR (nPCR) was carried out for further amplification and insertion of restriction sites into the PCR product via the primers in order to facilitate cloning in expression vectors.

(9) 5′ primers containing the BSSHII restriction site (capital letters) were used according to this configuration:

(10) 5′ primer BSSHII VH-FW1: attttttttGCGCGCtgt-(FW1-V-heavy family specific sequences); 3′ primers: the 3′ BsiWI Jκ-primers as published in Wardemann et al. (2003) were used.

(11) FIG. 3: Amplification of Linked Immunoglobulin Variable Heavy- and Light Chains From Encapsulated (Single) Antibody-Expressing Cells.

(12) A two step PCR protocol was used to amplify variable heavy- and light chains derived from a single cell via solid matrix-bound cDNA (oligo dT Dynabeads).

(13) (A) 1st PCR amplification of variable region of heavy and light chains followed by overlap extension in single tube/one step reaction on cDNA bound by oligo dT Dynabeads (lane 2). As control the same procedure was carried out using cells not expressing an antibody (lane 1).

(14) (B) 2nd (nested) PCR on 1st-round-PCR products showing the presence of a linked immunoglobulin heavy-and light chain derived from antibody expressing cells (lane 2) but not from control cells (lane 1).

(15) FIG. 4: Schematic Overview on Cloning Steps to Generate the Tri-Cistronic Antibody Expression Plasmid.

(16) I. Linked IgVH+IgVL PCR product is inserted into a shuttle vector providing the immunoglobulin signal peptide and the constant region of Ig kappa/lambda.

(17) II. A polynucleotide cassette providing the constant part of IgH, a cleavage site for later proteolytic cleavage and a signal peptide for secretion of Ig kappa/lambda is inserted by molecular cloning.

(18) III. The resulting bi-cistronic expression cassette is shuttled into a lentivector transfer vector by molecular cloning to yield the final Tri-cistronic expression vector used to generate lentivector particles.

(19) FIG. 5: Clonal Propagation of Expressor Cells in Nanoliter Reactors and Detection of Antibody Production.

(20) A) Clonal cell cluster (CCC) arising from a single CHO expressor cell eight days after encapsulation in a nanoliter reactor (NLR). The cell was transformed by a lentivector particle encoding the tri-cistronic antibody—EGFP-expression cassette prior to encapsulation.

(21) B) Production of antibody (full IgG) by clonal cell cluster (clone AB2). Western blot of Prot G purified antibody recovered from fluid inside NLR. PAGE was run under reducing conditions showing expression of Ig-heavy-and Ig-light chain.

(22) FIG. 6: High Throughput Single-Cell RT-PCR of Immunoglobulin Heavy and Light Chain mRNA.

(23) A) Droplets containing antibody expressing cells and mRNA capture beads before cell lysis (Step 1: cell encapsulation). Cells and oligo-dT Dynabeads in lysis buffer were encapsulated in Pico-Surf 2 emulsion oil, volume per droplet is approximately 1 nl, picture was taken directly after encapsulation and before cell lysis.

(24) B) Fluorescence activated sorting of singlet cDNA loaded capturing beads upstream of digital droplet PCR. Square gate defines singlet bead population.

(25) C) Single bead digital droplet PCR reveals the presence of a major population of capture beads containing both IgH and IgL, amplified sequences (dotted circle). Presence of IgH and IgL cDNA is monitored by release of a quenched fluorophore specific for either Ig chain by the PCR reaction (IgH=Y axis, IgL=X axis). Droplet PCR was analyzed using the QX100 droplet analyzer.

(26) D) Overlap extension PCR demonstrating the generation of linked IgH and IgL derived from single mRNA capturing beads. 1st PCR performed in emulsion droplets and second (nested) PCR performed in bulk.

(27) FIG. 7: Propagation of a NLR Culture Representing the Library of Antibody Expressor Cells

(28) A) Photo of NLR culture composed of single encapsulated expressor cells.

(29) B) Photo micrograph of a section of the bulk NLR culture at day 1.

(30) C) Fluorescent photo micrograph of NLR taken at day 4 of culture showing GFP positive clonal cell clusters (arrows) growing inside the NLRs that were the result of clonal expansion of a single encapsulated expressor cell.

(31) FIG. 8: Identification and Isolation of a Clonal Cell Line Expressing a mAb of Interest.

(32) A) 30 NLR bearing AB2 expressor cells were mixed to a more than 300 fold excess of NLRs bearing irrelevant expressor cells and bulk culture was plated in 80 wells of a 96 well plate. 4 days after encapsulation and plating the supernatant fluid of the NLR cultures was assayed by AB2-antigen-specific ELISA and AB2 positive culture wells were detected. Culture H5 was selected for cloning (arrow).

(33) B) NLR singularization and identification of clonal cell line E4. Single NLRs from bulk culture H5 were placed in individual culture wells. After 3 days, the supernatant fluid was assayed by AB2-ELISA and well E4 was identified demonstrating isolation of a clonal cell line that expressed an AB2-specific monoclonal antibody.

(34) Positive controls (+) were run in wells A1 and A2.

REFERENCES

(35) Embleton et al., Nucleic Acids Research, Vol. 20, No. 15, 3831 (1992)

(36) Wardemann, et al., Science 301, 1374 (2003)

(37) Boulianne et al., Nature 312, 643-646, (1984)

(38) Morrisson et al., PNAS 82, 6851-6855 (1984)

(39) Card et al., Cancer Immunol Immunother. April; 53(4):345-57 (2004)

(40) Szymczak et al., Nat Biotechnol 22:589-594 (2004)

(41) Serp et al., Biotechnology and Bioengineering, Vol. 70 (1) (2000)

(42) Gomez-Herreros et al., Nucleic Acids Research, 40, 6508-6519 (2012)

(43) Martinez et al. Macromol. Biosciences, 12, 946-951 (2012)

(44) Prüße et al., Chem. Eng Technol. 1998, 21(1):29-33

(45) McHeyzer-Williams and Ahmed, Curr. Opin. Immunol. 11, 172-179, (1999)

(46) Bernasconi et al., Science 298, 2199-202 (2002)

(47) Meijer et al., J Mol Biol. 358: 764-772 (2006)

(48) Dull et al., J. Virol 72(11) (1998)

(49) Diehl et al. Nature Methods, 3, 551-559 (2006)

(50) Primers Used

(51) First PCR:

(52) Ig-variable heavy chain:

(53) TABLE-US-00004 Forward primers: 1. 5′ SP-VH1 ACAGGTGCCCACTCCCAGGTGCAG 2. 5′ SP-VH3 AAGGTGTCCAGTGTGARGTGCAG 3. 5′ SP-VH4/6 CCCAGATGGGTCCTGTCCCAGGTGCAG 4. 5′ SP-VH5 CAAGGAGTCTGTTCCGAGGTGCAG Reverse primer: 5. 3′CHg1 adaptor-GTTGTCCACCTTGGTGTTGCTGG 6. 3′ Cμ CH1 adaptor-GGGAATTCTCAGAGGAGACGA

(54) Ig-variable kappa chain:

(55) TABLE-US-00005 Forward primers: 7. 5′ SP Vκ1/2 reverseadaptor- ATGAGGSTCCCYGCTCAGCTGCTGG 8. 5′ SP Vκ3 reverseadaptor- CTCTTCCTCCTGCTACTCTGGCTCCCAG 9. 5′ SP Vκ4 reverseadaptor- ATTTCTCTGTTGCTCTGGATCTCTG Reverse primer: 10. 3′ Cκ 543 GTTTCTCGTAGTCTGCTTTGCTCA

(56) Ig-variable lambda chain:

(57) TABLE-US-00006 Forward primers: 11. 5′ SP Vλ1 reverseadaptor- GGTCCTGGGCCCAGTCTGTGCTG 12. 5′ SP Vλ2 reverseadaptor- GGTCCTGGGCCCAGTCTGCCCTG 13. 5′ SP Vλ3 reverseadaptor- GCTCTGTGACCTCCTATGAGCTG 14. 5′ SP Vλ4/5 reverseadaptor- GGTCTCTCTCSCAGCYTGTGCTG 15. 5′ SP Vλ6 reverseadaptor- GTTCTTGGGCCAATTTTATGCTG 16. 5′ SP Vλ7 reverseadaptor- GGTCCAATTCYCAGGCTGTGGTG 17. 5′ SP Vλ8 reverseadaptor- GAGTGGATTCTCAGACTGTGGTG Reverse primer: 18. 3′ Cλ CACCAGTGTGGCCTTGTTGGCTTG

(58) Overlap Extension PCR:

(59) TABLE-US-00007 Forward primers: 19. 5′ BSSH2 VH1/5 CTGCAGCGCGCGTACAT TCCGAGGTGCAGCT GGTGCAG 20. 5′ BSSH2 VH3 CTGCAGCGCGCGTACATTCTGAGGTGCAGCTGG TGGAG 21. 5′ BSSH2 VH4 CTGCAGCGCGCGTACATTCCCAGGTGCAGCTGC AGGAG 22. 5′ BSSH2 VH3-23 CTGCAGCGCGCGTACATTCTGAGGTGCAGCT GTTGGAG 23. 5′ BSSH2 VH4-34 CTGCAGCGCGCGTACATTCCCAGGTGCAGCT ACAGCAGTG Reverse primers: 24. 3′BsiWI Jκ1/2/4 GCCACCGTACGTTTGATYTCCACCTTGGTC 25. 3′BsiWI Jκ3 GCCACCGTACGTTTGATATCCACTTTGGTC 26. 3′XhoICλ CTCCTCACTCGAGGGYGGGAACAGAGTG

(60) oePCR Linker Primers

(61) TABLE-US-00008 Ig-variable heavy chain: 28. 3′ SaiI JH tattcgcactgcgcggcGTCGACgc-(JH family specific sequences) Ig-variable light chain: 29. 5′ XbaI Vk gccgcgcagtgcgaataTCTAGAtgt-(FW1-V-kappa family specific sequences) 30. 5′ XbaI Vl gccgcgcagtgcgaataTCTAGAtgt-(FW1-V- lambda family specific sequences)

(62) nPCR

(63) TABLE-US-00009 5′ primers: BSSHII VH-FW1 attttttttGCGCGCtgt-(FW1-V-heavy family specific sequences 3′ primers 3′ BsiWI Jκ-primers (see table below)

(64) TABLE-US-00010 TABLE 1 further primers to be used to carry out the invention. Restriction sites are shown in bold. Sense Antisense IgH First PCR 5′LVH1 ACAGGTGCCCACTCCCAGGTGCAG 3′ Cμ CH1 GGGAATTCTCAGAGGAGACGA 5′ L-VH3 AAGGTGTCCAGTGTGARGTGCAG 5′ L-VH4/6 CCCAGATGGGTCCTGTCCCAGGTGCAG 5′ L-VH5 CAAGGAGTCTGTTCCGAGGTGCAG Second PCR 5′ Agel VH1/5 CTGCAACCGGTGTACATTCCGAGGT 3′ SalI JH1/2 TGCGAAGTCGACGCCTGAGGAG GCAGCTGGTGCAG ACGGTGACCAG 5′ Agel VH3 CTGCAACCGGTGTACATTCTGAGGT 3′ SalI JH3 TGCGAAGTCGACGCTGAAGAGA GCAGCTGGTGGAG CGGTGACCATTG 5′ Agel VH4 CTGCAACCGGTGTACATTCCCAGGT 3′ SalI JH4/5 TGCGAAGTCGACGCCTGAGGAG GCAGCTGCAGGAG ACGGTGACCAG 5′ Agel VH3-23 CTGCAACCGGTGTACATTCTGAGGT 3′ SalI JH6 TGCGAAGTCGACGCTGAGGAGA CAGCGCTGTTGGAG CGGTGACCGTG 5′ Agel VH3-34 CTGCAACCGGTGTACATTCCCAGGT GCAGCTACAGCAGTG Igλ First PCR L 5′ Vλ1 GGTCCTGGGCCCAGTCTGTGCTG 3′ Cλ CACCAGTGTGGCCTTGTTGGCTTG 5′ L Vλ2 GGTGCTGGGCCCAGTCTGCCCTG 5′ L Vλ3 GCTCTGTGACCTCCTATGAGCTG 5′ L Vλ4/5 GGTCTCTCTCSCAGCYTGTGCTG 5′ L Vλ6 GTTCTTGGGCCAATTTTATGCTG 5′ L Vλ7 GGTCCAATTCYCAGGCTGTGGTG 5′ L Vλ8 GAGTGGATTCTCAGACTGTGGTG Second PCR 5′ Agel Vλ1 CTGCTACCGGTTCCTGGGCCCAGTC 3′XhoI Cλ CTCCTCACTCGAGGYGGGAACA TGTGCTGACKCAG GAGTG 5′ Agel Vλ2 CTGCTACCGGTTCCTGGGCCCAGTC TGCCCTGACTCAG 5′ Agel Vλ3 CTGCTACCGGTTCTGTGACCTCCTA TGAGCTGACWCAG 5′ Agel Vλ4/5 CTGCTACCGGTTCTCTCTCSCAGCYT GTGCTGACTCA 5′ Agel Vλ6 CTGCTACCGGTTCTTGGGCCAATTTT ATGCTGACTCAG 5′ Agel Vλ7/8 CTGCTACCGGTTCCAATTCYCAGRCT GTGGTGACYCAG Igκ First PCR 5′ L Vκ1/2 ATGAGGSTCCCYGCTCAGCTGCTGG 3′ Cκ 543 GTTTCTCGTAGTCTGCITTGCTCA 5′ L Vκ3 CTCTTCCTCCTGCTACTCTGGCTCCCAG 5′ L Vκ4 ATTTCTCTGTTGCTCTGGATCTCTG Second PCR 5′ Pan Vκ ATGACCCAGWCTCCABYCWCCCTG 3′ Cκ 494 GTGCTGTCCTTGCTGTCCTGCTC Specific PCR 5′ Agel Vκ 1-5 CTGCAACCGGTGTACATTCTGACAT 3′ BsiWI Jκ1/2/4 GCCACCGTACGTTTGATYTCCAC CCAGATGACAGTC CTTGGTC 5′ Agel Vκ 1-9 TTGTGCTGCAACCGGTGTACATTCA 3′ BsiWI Jκ3 GCCACCGTACGTTTGATATCCAC GACATCCAGTTGACCCAGTCT TTTGGTC 5′ Agel Vκ 1D-43 CTGCAACCGGTGTACATTGTGCCAT 3′BsiWI Jκ5 GCCACCGTACGTTTAATCTCCA CCGGATGACCCAGTC GTCGTGTC 5′ Agel Vκ 2-24 CTGCAACCGGTGTACATGGGGATAT TGTGATGACCCAGAC 5′ Agel Vκ 2-28 CTGCAACCGGTGTACATGGGGATA TTGTGATGACTCAGTC 5′ Agel Vκ 3-11 TTGTGCTGCAACCGGTGTACAITC AGAAATTC 5′ Agel Vκ 3-15 CTGCAACCGGTGTACATTCAGAAAT AGTGATGACGCAGTC 5′ Agel Vκ 3-20 TTGTGCTGCAACCGGTGTACATTCA GAAATTGTGTTGACGCAGTCT 5′ Agel Vκ 4-1 CTGCAACCGGTGTACATTCGGACAT CGTGATGACCCAGTC