We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.
Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.
The method described here allows the rapid and versatile production of fully human monoclonal antibodies (mAbs) against a desired antigen (Ag). mAbs are essential tools in many fundamental research applications in vitro and in vivo: flow cytometry, histology, western-blotting, and blocking experiments for example. Furthermore, mAbs are being used more and more in medicine to treat autoimmune diseases, cancer, and to control transplantation rejection1. For example, anti-CTLA-4 and anti-PD-1 (or anti-PD-L1) mAbs were recently used as immune checkpoint inhibitors in cancer treatments2.
The first mAbs were produced by immunoglobulin (Ig)-secreting hybridomas obtained from the splenic cells of immunized mice or rats. However, the strong immune response against murine or rat mAbs hampers their therapeutic use in humans, due to their rapid clearance and the probable induction of hypersensitivity reactions3. To tackle this problem, animal protein sequences of mAbs have been partially replaced by human ones to generate so-called chimeric mouse-human or humanized antibodies. However, this strategy only partially decreases immunogenicity, while substantially increasing both the cost and the time-scale of production. A better solution is to generate human mAbs directly from human B cells and several strategies for this are available. One of them is the use of phage or yeast display. This involves displaying variable domains from a combinatorial library of random human Ig heavy and light chains on phages or yeasts, and carrying out a selection step using the specific antigen of interest. A major drawback of this strategy is that heavy and light chains are randomly associated, leading to a very large increase in the diversity of generated antibodies. Antibodies obtained are unlikely to correspond to those that would arise from a natural immune response against a particular Ag. Moreover, human protein folding and post-translational modifications are not systematically reproduced in prokaryotes or even in yeasts. A second human mAb production method is the immortalization of natural human B cells, by Epstein-Barr virus infection or expression of the anti-apoptotic factors BCL-6 and BCL-XL4. However, this method is applicable only to memory B cells and is inefficient, requiring screening of numerous mAb-producing immortalized B cells to identify the few (if any) mAb clones with the desired antigenic specificity. The method is thus both costly and time consuming.
A new protocol has recently been described for production of human mAbs from isolated single B cells5. It relies on an optimized single-cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) for amplification of both the heavy- and light-chain encoding segments from a single sorted B cell. This is followed by the cloning and expression of these segments in a eukaryotic expression system, thus allowing reconstruction of a fully human mAb. This protocol has been used successfully starting from B cells from vaccinated donors. Cells were harvested several weeks after vaccination to obtain higher frequencies of B cells directed against the desired Ag, and thus limit the time required for screening6. Other fully human mAbs have also been produced from HIV+ (Human Immunodeficiency Virus) infected patients7 and melanoma patients8. Despite these advances, there is still no procedure available that enables the isolation of Ag-specific B cells independent of their memory phenotype or frequency.
The procedure described here leads to efficient ex vivo isolation of human circulating B cells based on their BCR specificity, followed by the production of fully human antigen-specific mAbs in high yield and with a low screening time. The method is not restricted to memory B cells or antibody-secreting B cells induced after an immune response, but can also be applied to the human naïve B cell repertoire. That it works even starting from Ag-specific B cells present at very low frequencies is a good indication of its efficiency. The principle of the method is as follows: Peripheral Blood Mononuclear Cells (PBMC) are stained with two tetramers presenting the antigen of interest, each labeled with a different fluorochrome (e.g., Phycoerythrin (PE) and Allophycocyanin (APC)), and a third tetramer presenting a closely related antigen conjugated with a third fluorochrome (e.g., Brillant Violet 421 (BV421)). To enrich for antigen-binding cells, cells are then incubated with beads coated with anti-PE and anti-APC Abs, and sorted in cell separation columns. The PE+ APC+ cell fraction is selected, stained with a variety of mAbs specific for different PBMC cell types to permit identification of B cells, and subjected to flow cytometry cell sorting. B cells which are PE+ and APC+, but Brilliant Violet–, are isolated. This step counter-selects cells which are not B cells or do not bind to the tetramerized antigen, but do bind to either PE or APC (these cells will be PE+ APC– or PE– APC+) or to the non-antigen part of the tetramers used (these cells will be BV421+). B cells not highly specific for the epitope of interest are also counter-selected at this step (these cells will also be BV421+). Thus, this method can purify highly specific B cells expressing B-cell Receptors (BCRs) able to discriminate between two very closely related antigens. Single specific B cells are collected in tubes and their PCR-amplified Ig cDNAs (complementary deoxyribonucleic acids) cloned and expressed by a human cell line as secreted IgG mAbs.
As a proof of concept, this study describes the efficient generation of human mAbs, which recognize a peptide presented by a major histocompatibility complex class I (MHC-I) molecule and can discriminate between this peptide and other peptides loaded on the same MHC-I allele. Although the level of complexity of this Ag is important, this method allows (i) high yield recovery of Ag-specific mAbs; (ii) production of mAbs able to discriminate between two structurally close Ags. This approach can be extended to vaccinated or infected patients without any protocol modification, and has also already been successfully implemented in a humanized rat system9. Thus, this study describes a versatile and efficient approach to generate fully human mAbs that can be used in basic research and immunotherapy.
The proposed protocol is a powerful method for the generation of human mAbs directly from Ag-specific B cells circulating in the blood. It combines three important aspects: (i) the use of a tetramer-associated magnetic enrichment, which allows an ex vivo isolation of even rare Ag-binding B cells; (ii) a gating strategy that uses three Ag tetramers (two relevant ones and one irrelevant one) labelled with three different fluorochromes to isolate, by flow cytometry, only the B cells expressing a BCR specific for th…
The authors have nothing to disclose.
We thank the Cytometry Facility "CytoCell" (SFR Santé, Biogenouest, Nantes) for expert technical assistance. We thank also all the staff of recombinant protein production (P2R) and of IMPACT platforms (INSERM 1232, SFR Santé, Biogenouest, Nantes) for their technical support. We thank Emmanuel Scotet and Richard Breathnach for constructive comments on the manuscript. This work was financially supported by the IHU-Cesti project funded by the « Investissements d'Avenir » French Government program, managed by the French National Research Agency (ANR) (ANR-10-IBHU-005). The IHU-Cesti project is also supported by Nantes Métropole and Région Pays de la Loire. This work was realized in the context of the LabEX IGO program supported by the National Research Agency via the investment of the future program ANR-11-LABX-0016-01.
HEK 293A cell line | Thermo Fisher scientific | R70507 | |
DMEM (1X) Dulbecco's Modified Eagle Medium | Gibco by life technologies | 21969-035 | (+) 4,5g/L D-Glucose 0,11g/L Sodium Pyruvate (-) L-Glutmine |
RPMI medium1640 (1X) | Gibco by life technologies | 31870-025 | |
Bovine Serum Albumine (BSA) | PAA | K45-001 | |
Nutridoma-SP | Roche | 11011375001 | 100X Conc |
PBS-Phosphate Buffered Saline (10X) pH 7,4 | Ambion | AM9624 | |
EDTA (Ethylenediaminetetraacetic acid) 0,5M pH=8 | Invitrogen by Life Technologies | 15575-020 | |
Fetal Bovine serum (FBS) | Dominique Dutscher | S1810-500 | |
Ficoll – lymphocytes separation medium | EuroBio | CMSMSL01-01 | density 1,0777+/-0,001 |
streptavidin R-phycoerythrin conjugate | Invitrogen by Life Technologies | S21388 | premiun grade 1mg/ml contains 5mM sodium azide |
Streptavidin, allophycocyanin conjugate | Invitrogen by thermoFisher scientific | S32362 | 1mg/ml 2mM azide premium grade |
Brilliant violet 421 streptavidin | Biolegend | 405225 | conc : 0,5mg/ml |
Anti-PE conjugated magnetic MicroBeads | Miltenyi Biotec | 130-048-801 | |
Anti-APC conjugated magnetic MicroBeads | Miltenyi Biotec | 130-090-855 | |
MidiMACs or QuadroMACS separotor | Miltenyi Biotec | 130-042-302/130-090-976 | |
LS Columns | Miltenyi Biotec | 130-042-401 | |
CD3 BV510 BD horizon | BD Pharmingen / BD Biosciences | 563109 | Used dilution 1:20 |
CD19 FITC | BD Pharmingen / BD Biosciences | 345788 | Used dilution 1:20 |
CD14 PerCPCy5.5 | BD Pharmingen / BD Biosciences | 561116 | Used dilution 1:50 |
CD16 PerCPCy5.5 | BD Pharmingen / BD Biosciences | 338440 | Used dilution 1:50 |
7AAD | BD Pharmingen / BD Biosciences | 51-68981E (559925) | Used dilution 1:1000 |
FACS ARIA III Cell Sorter Cytometer | BD Biosciences | ||
8-strip PCR tubes | Axygen | 321-10-061 | |
Racks for 96 microtubes | Dominique Dutscher | 45476 | |
RNAseOUT Ribonuclease Inhibitor (recombinant) | Invitrogen by thermoFisher scientific | 10777-019 | qty:5000U (40U/ul) |
Distilled Water Dnase/Rnase Free | Gibco | 10977-035 | |
Oligod(T)18 mRNA Primer | New England BioLabs | S1316S | 5.0 A260unit |
Random hexamers | Invitrogen by thermoFisher scientific | N8080127 | qty : 50uM, 5nmoles |
Superscript III Reverse transcriptase | Invitrogen by thermoFisher scientific | 18080-044 | qty : 10000U (200U/ul) |
GoTaq G2 Flexi DNA polymerase | Promega | M7805 | |
dNTP Set, Molecular biology grade | Thermo Scientific | R0182 | 4*100umol |
5LVH1 | Eurofins | ACAGGTGCCCACT CCCAGGTGCAG |
First round of PCR – Amplification of heavy chains – Outer primers – Forward Prmers |
5LVH3 | Eurofins | AAGGTGTCCAGTG TGARGTGCAG |
First round of PCR – Amplification of heavy chains – Outer primers – Forward Prmers |
5LVL4_6 | Eurofins | CCCAGATGGGTCC TGTCCCAGGTGCAG |
First round of PCR – Amplification of heavy chains – Outer primers – Forward Prmers |
5LVH5 | Eurofins | CAAGGAGTCTGTT CCGAGGTGCAG |
First round of PCR – Amplification of heavy chains – Outer primers – Forward Prmers |
3HuIgG_const_anti | Eurofins | TCTTGTCCACCTT GGTGTTGCT |
First round of PCR – Amplification of heavy chains – Outer primers -Reverse primers for human Ig- Bacteria PCR screening |
3CuCH1 | Eurofins | GGGAATTCTCACA GGAGACGA |
First round of PCR – Amplification of heavy chains – Outer primers -Reverse primers for human Ig |
5AgeIVH1_5_7 | Eurofins | CTGCAACCGGTGTACATTCC GAGGTGCAGCTGGTGCAG |
Second round of PCR – Amplification of heavy chains – Inner primers -Forward primers |
5AgeIVH3 | Eurofins | CTGCAACCGGTGTACATTCT GAGGTGCAGCTGGTGGAG |
Second round of PCR – Amplification of heavy chains – Inner primers -Forward primers |
5AgeIVH3_23 | Eurofins | CTGCAACCGGTGTACATTCT GAGGTGCAGCTGTTGGAG |
Second round of PCR – Amplification of heavy chains – Inner primers -Forward primers |
5AgeIVH4 | Eurofins | CTGCAACCGGTGTACATTCC CAGGTGCAGCTGCAGGAG |
Second round of PCR – Amplification of heavy chains – Inner primers -Forward primers |
5AgeIVH4_34 | Eurofins | CTGCAACCGGTGTACATTCC CAGGTGCAGCTACAGCAGTG |
Second round of PCR – Amplification of heavy chains – Inner primers -Forward primers |
5AgeIVH1_18 | Eurofins | CTGCAACCGGTGTACATTCC CAGGTTCAGCTGGTGCAG |
Second round of PCR – Amplification of heavy chains – Inner primers -Forward primers |
5AgeIVH1_24 | Eurofins | CTGCAACCGGTGTACATTCC CAGGTCCAGCTGGTACAG |
Second round of PCR – Amplification of heavy chains – Inner primers -Forward primers |
5AgeIVH3__9_30_33 | Eurofins | CTGCAACCGGTGTACATTCT GAAGTGCAGCTGGTGGAG |
Second round of PCR – Amplification of heavy chains – Inner primers -Forward primers |
5AgeIVH6_1 | Eurofins | CTGCAACCGGTGTACATTCC CAGGTACAGCTGCAGCAG |
Second round of PCR – Amplification of heavy chains – Inner primers -Forward primers |
3SalIJH1_2_4_5 | Eurofins | TGCGAAGTCGACG CTGAGGAGACGGTGACCAG |
Second round of PCR – Amplification of heavy chains – Inner primers -Reverse primers |
3SalIJH3 | Eurofins | TGCGAAGTCGACG CTGAAGAGACGGTGACCATTG |
Second round of PCR – Amplification of heavy chains – Inner primers -Reverse primers |
3SalIJH6 | Eurofins | TGCGAAGTCGACG CTGAGGAGACGGTGACCGTG |
Second round of PCR – Amplification of heavy chains – Inner primers -Reverse primers |
5'LVk1_2 | Eurofins | ATGAGGSTCCCYG CTCAGCTGCTGG |
First round of PCR – Amplification of light chains k – Outer primers -Forward primers |
5'LVk3 | Eurofins | CTCTTCCTCCTGC TACTCTGGCTCCCAG |
First round of PCR – Amplification of light chains k – Outer primers -Forward primers |
5'LVk4 | Eurofins | ATTTCTCTGTTGC TCTGGATCTCTG |
First round of PCR – Amplification of light chains k – Outer primers -Forward primers |
3'Ck543_566 | Eurofins | GTTTCTCGTAGTC TGCTTTGCTCA |
First round of PCR – Amplification of light chains k – Outer primers -Reverse primers- Bacteria PCR screening |
5'AgeIVk1 | Eurofins | CTGCAACCGGTGTACATTCT GACATCCAGATGACCCAGTC |
Second round of PCR – Amplification of light chains k – Inner primers -Forward primers |
5'AgeIVk1_9_1–13 | Eurofins | TTGTGCTGCAACCGGTGTAC ATTCAGACATCCAGTTGACCCAGTCT |
Second round of PCR – Amplification of light chains k – Inner primers -Forward primers |
5'AgeIVk1D_43_1_8 | Eurofins | CTGCAACCGGTGTACATTGT GCCATCCGGATGACCCAGTC |
Second round of PCR – Amplification of light chains k – Inner primers -Forward primers |
5'AgeIVk2 | Eurofins | CTGCAACCGGTGTACATGGG GATATTGTGATGACCCAGAC |
Second round of PCR – Amplification of light chains k – Inner primers -Forward primers |
5'AgeIVk2_28_2_30 | Eurofins | CTGCAACCGGTGTACATGGG GATATTGTGATGACTCAGTC |
Second round of PCR – Amplification of light chains k – Inner primers -Forward primers |
5'AgeVk3_11_3D_11 | Eurofins | TTGTGCTGCAACCGGTGTAC ATTCAGAAATTGTGTTGACACAGTC |
Second round of PCR – Amplification of light chains k – Inner primers -Forward primers |
5'AgeVk3_15_3D_15 | Eurofins | CTGCAACCGGTGTACATTCA GAAATAGTGATGACGCAGTC |
Second round of PCR – Amplification of light chains k – Inner primers -Forward primers |
5'AgeVk3_20_3D_20 | Eurofins | TTGTGCTGCAACCGGTGTAC ATTCAGAAATTGTGTTGACGCAGTCT |
Second round of PCR – Amplification of light chains k – Inner primers -Forward primers |
5'AgeVk4_1 | Eurofins | CTGCAACCGGTGTACATTCG GACATCGTGATGACCCAGTC |
Second round of PCR – Amplification of light chains k – Inner primers -Forward primers |
3'BsiWIJk1_2_4 | Eurofins | GCCACCGTACGTT TGATYTCCACCTTGGTC |
Second round of PCR – Amplification of light chains k – Inner primers -Forward primers |
3'BsiWIJk3 | Eurofins | GCCACCGTACGTT TGATATCCACTTTGGTC |
Second round of PCR – Amplification of light chains k – Inner primers -Forward primers |
3'BsiWIJk5 | Eurofins | GCCACCGTACGTT TAATCTCCAGTCGTGTC |
Second round of PCR – Amplification of light chains k – Inner primers -Forward primers |
5'LVl1 | Eurofins | GGTCCTGGGCCCA GTCTGTGCTG |
First round of PCR – Amplification of light chains λ – Outer primers -Forward primers |
5'LVl2 | Eurofins | GGTCCTGGGCCCA GTCTGCCCTG |
First round of PCR – Amplification of light chains λ – Outer primers -Forward primers |
5'LVl3 | Eurofins | GCTCTGTGACCTC CTATGAGCTG |
First round of PCR – Amplification of light chains λ – Outer primers -Forward primers |
5'LVl4_5 | Eurofins | GGTCTCTCTCSCA GCYTGTGCTG |
First round of PCR – Amplification of light chains λ – Outer primers -Forward primers |
5'LVl6 | Eurofins | GTTCTTGGGCCAA TTTTATGCTG |
First round of PCR – Amplification of light chains λ – Outer primers -Forward primers |
5'LVl7 | Eurofins | GGTCCAATTCYCA GGCTGTGGTG |
First round of PCR – Amplification of light chains λ – Outer primers -Forward primers |
5LVl8 | Eurofins | GAGTGGATTCTCA GACTGTGGTG |
First round of PCR – Amplification of light chains λ – Outer primers -Forward primers |
3'Cl | Eurofins | CACCAGTGTGGCC TTGTTGGCTTG |
First round of PCR – Amplification of light chains λ – Outer primers -Forward primers |
5'AgeIVl1 | Eurofins | CTGCTACCGGTTCCTGGGCC CAGTCTGTGCTGACKCAG |
Second round of PCR – Amplification of light chains λ – Inner primers -forward primers |
5'AgeIVl2 | Eurofins | CTGCTACCGGTTCCTGGGCC CAGTCTGCCCTGACTCAG |
Second round of PCR – Amplification of light chains λ – Inner primers -forward primers |
5'AgeIVl3 | Eurofins | CTGCTACCGGTTCTGTGACC TCCTATGAGCTGACWCAG |
Second round of PCR – Amplification of light chains λ – Inner primers -forward primers |
5'AgeIVl4_5 | Eurofins | CTGCTACCGGTTCTCTCTCS CAGCYTGTGCTGACTCA |
Second round of PCR – Amplification of light chains λ – Inner primers -forward primers |
5'AgeIVl6 | Eurofins | CTGCTACCGGTTCTTGGGCC AATTTTATGCTGACTCAG |
Second round of PCR – Amplification of light chains λ – Inner primers -forward primers |
5'AgeIVl8 | Eurofins | CTGCTACCGGTTCCAATTCY CAGRCTGTGGTGACYCAG |
Second round of PCR – Amplification of light chains λ – Inner primers -forward primers |
3'XhoICl | Eurofins | CTCCTCACTCGAG GGYGGGAACAGAGTG |
Second round of PCR – Amplification of light chains λ – Inner primers -Reverse primers – Bacteria PCR screening |
Ab-vec-sense | Eurofins | GCTTCGTTAGAAC GCGGCTAC |
Bacteria PCR screening |
QA Agarose-TM, Molecular Biology Grade | MP Bio | AGAH0500 | |
NucleoFast 96 PCR Plate | Macherey Nagel | 743.100.100 | |
Enzyme Age I HF | New England Biolabs | R3552L | 20000U/ml |
Enzyme SalI HF | New England Biolabs | R3138L | 20000U/ml |
Enzyme Xho I | New England Biolabs | R0146L | 20000U/ml |
Enzyme BSIWI | New England Biolabs | R0553L | 10000U/ml |
HCg1 (Genbank accession number FJ475055) | |||
LCk (Genbank accession number FJ475056 ) | |||
LCl (Genbank accession number FJ517647) | |||
T4 DNA ligase | Invitrogen by thermoFisher scientific | 15224.017 | 100U (1U/ul) |
2X YT medium | Sigma Aldrich | Y1003-500ML | |
Ampicillin | Sigma Aldrich | 10835242001 | |
LB (Luria Bertani) Broth (Lennox) | Sigma Aldrich | L3022-250G | |
Nucleospin Plasmid DNA, RNA and protein purification | Macherey Nagel | 740588.250 | |
Jet PEI DNA transfection reagent | PolyPlus | 101-40 | |
Flat bottom96-well plate | Falcon | 353072 | |
V-bottom 96-well plate | Nunc/Thermofisher | 055142 | |
Nunc easy 175 cm2 flasks | Nunc/Thermofisher | 12-562-000 | |
ELISA/ELISPOT coating buffer | eBiosciences | 00-0044-59 | |
Nunc maxisorp flat bottom 96 well ELISA plates | Nunc/Thermofisher | 44-2404-21 | high protein binding |
Anti-human IgG Ab conjugated to horseradish peroxidase (HRP) | BD Pharmingen / BD Biosciences | 55788 | |
TMB substrate | BD Biosciences | 555214 | |
Streptavidin | Sigma | S0677 | |
1 mL-HiTrap protein A HP column | GE Healthcare | 17-0402-01 | |
ÄKTA FPLC | GE Healthcare | 18190026 | |
Superdex 200 10/300 GL column | GE Healthcare | 17517501 | |
NGC Quest 10 Plus Chromatography System | BioRad | 7880003 |