Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

All human peripheral blood samples were obtained from anonymous adult donors after informed consent, in accordance with the local ethics committee (Etablissement Français du Sang, EFS, Nantes, procedure PLER NTS-2016-08).

1. Isolation of Human Peripheral Blood Mononuclear Cells

NOTE: Starting material can be total human peripheral blood or cytapheresis samples. Samples should not be older than 8 h and supplemented with anticoagulants (e.g., heparin).

1. Dilute blood with 2 volumes (human peripheral blood) or 5 volumes (cytapheresis sample) of RPMI (Roswell Park Memorial Institute) medium.
2. Carefully layer 35 mL of diluted cell suspension onto 15 mL of density gradient medium in a 50-mL conical tube. Make as many tubes as necessary to distribute all the diluted blood. Centrifuge at 1,290 x g for 25 min at room temperature in a swinging-bucket rotor with brake off.
3. Carefully aspirate the mononuclear cell layer at the interface between the density gradient medium and the plasma layer, and transfer the cells to a new 50 mL conical tube.
4. Fill the tube with RPMI medium, mix and centrifuge at 1,290 x g for 10 min at room temperature. Carefully remove the supernatant.
5. Resuspend the cell pellet in 20 mL of RPMI medium and centrifuge at 200 x g for 10 min to remove the platelets. Carefully remove the supernatant. Repeat this step once.
6. Resuspend the cell pellet in RPMI medium with 10% Fetal Bovine Serum (FBS).
   NOTE: Proceed directly to tetramer labeling or keep cells at 4 °C overnight at a concentration of 10⁷ cells/mL.

2. Tetramer-associated Magnetic Enrichment of Ag-specific B Cells

1. Prepare Ag-tetramers by adding either PE or APC-labeled premium grade streptavidin or BV421-labeled streptavidin at a molar ratio of 1:4 to biotinylated antigen monomers. Add the appropriate amount of streptavidin-conjugate in three separate portions, adding one aliquot every 10 min at room temperature.
2. Distribute cells (up to 3 x 10⁸ per tube) in 15 mL conical tubes and centrifuge at 460 x g for 5 min.
   NOTE: The following protocol is for one tube.
3. Resuspend the cell pellet in 200 µL of PBS (Phosphate Buffered Saline) with 2% FBS. Add PE-, APC-, and BV421-conjugated tetramers, each to a final concentration of 10 µg/mL. Mix well and incubate for 30 min at room temperature.
4. Prepare ice-cold cell separation buffer (SB) using PBS with 0.5% BSA (Bovine Serum Albumin) and EDTA (Ethylenediaminetetraacetic acid) 2 mM.
5. Add 10 mL of SB. Pellet cells by centrifugation at 460 x g for 5 min.
6. Resuspend cells in 500 µL of ice-cold SB. Add 50 µL of anti-PE and 50 µL of anti-APC magnetic microbeads.
7. Mix well and incubate for 20 min at 4 °C.
8. Repeat step 2.5 twice.
9. Eliminate the supernatant carefully andresuspend the cell pellet at a concentration of 2 x 10⁸ cells/mL in ice cold SB.
10. Equilibrate a large magnetic column positioned on a magnet with 3 mL of SB.
11. Transfer cell suspension from 2.9 onto the top of the equilibrated column and allow it to drain completely. CRITICAL STEP: To avoid clumping of the column, pass cells through a 70 µm nylon mesh.
12. Rinse the tube which contained the cells with 3 mL of ice cold SB and transfer the buffer directly onto the column (in case of filtration, rinse the 70 µm nylon mesh).
13. When the buffer has completely drained into the column, add another 3 mL of ice cold SB to the column.
14. Repeat step 2.13 for another 3 mL wash.
15. Remove the column from the magnet and place over a 15-mL conical collecting tube.
16. Add 5 mL of ice cold SB onto the top of the column and immediately flush the cells out of the column using the plunger. Repeat this step once.
17. Centrifuge the collected cells (enriched relevant tetramer positive cell fraction) at 460 x g for 5 min and proceed to additional antibody staining.
    NOTE: Pool collected cells from all tubes, if appropriate.

3. Staining of Ag-specific Human B Cells and Cell Sorting

1. Resuspend the cell pellet with a cocktail of anti-human antibodies against CD3 (final dilution: 1:20), CD19 (1:20), CD14 (1:50), CD16 (1:50), and 7AAD (1:1000) in a final volume of 100 µL of PBS with 2% FBS. Incubate for 30 min at 4 °C.
2. Add 10 mL of PBS to cells, centrifuge at 460 x g for 5 min, and eliminate the supernatant. Repeat this step once. Resuspend the cell pellet in 200 µL of PBS, filter onto 70 µm nylon mesh.
3. Proceed to sort specific B cells at the single cell level on a cell sorter cytometer with a 100 µm-nozzle and a pressure of 20 psi without exceeding 1,000 events/s⁹.
   NOTE: The gating strategy used is shown in Figure 1. Cells were first gated on CD14^–CD16^–7AAD^– cells to exclude monocytes, NK, and dead cells (not shown in Figure 1). Then CD19⁺ B cells were selected before gating on double stained cells by PE and APC relevant Ag-tetramers. Non-specific B cells were excluded by gating on BV421 negative cells (unstained by irrelevant Ag-tetramer).
4. Collect single B cells into 8-strip PCR tubes previously filled with 10 µL of 1x PBS and 10 units of RNAse Inhibitor and placed on a rack for 96 microtubes.Immediately freeze the PCR tubes at -80 °C.
   NOTE: The sort masks chosen on the cytometer instrument are yield mask: 0, purity mask: 32, and phase mask: 0. This single-cell deposit could be adjusted to a 96- or 384-well PCR plate if needed. Single B cells must be frozen as quickly as possible and can be left at -80 °C for several weeks, if needed.

4. Single Cell RT-PCR

1. Lyse cells by directly heating the frozen samples in PCR strips at 70 °C for 5 min in a dry bath.
2. Transfer strips to ice and proceed to reverse transcription (RT) by adding 10 µL per tube of a 2x master mix buffer containing 1 mM dNTP, 25 µg/mL oligod(T) primers, 5 µM random hexamers, 20 units of RNAse inhibitor, and 400 units of reverse transcriptase.
   NOTE: After cell thawing, RT must be performed quickly to avoid RNA degradation.
3. Incubate at 25 °C for 5 min to allow random hexamers to hybridize, then incubate for 1 h at 50 °C, and finish with an incubation at 95 °C for 3 min to stop the reaction.
4. Proceed with two rounds of nested PCR amplification for each of the regions encoding for variable heavy (vH) and kappa (vLκ) or lambda light chains (vLλ).
   NOTE: Alternatively, RT samples can be frozen at -20 °C and kept for one week.
   1. For the first round of PCR, add 3 µL of cDNA for a 40 µL final volume containing 1.5 mM MgCl₂, 0.25 mM dNTPs, 2.5 units of DNA Polymerase, and 200 nM of outer primers (see Figure 2 and Table of Materials for primer sequences).
      NOTE: Composition of outer primers mix. For heavy chain amplification: 4 forward primers (5'LVH mix) and 2 reverse primers (3'CµCγ mix). For light kappa chain amplification, 3 forward primers (5'LV| mix) and 1 reverse primer (3'Cκ543-566). For light lambda chain amplification, 7 forward primers (5'LVl mix) and 1 reverse primer (3'Cl).
      NOTE: Primers were designed by Tiller et al. for amplification of all the heavy and light chain family genes⁵.
   2. Apply the following cycling conditions: 94 °C for 4 min, followed by 40 cycles of 30 s at 94 °C, 30 s at 58 °C for VH and VLκ (60 °C for VLλ), and 55 s at 72 °C, with a final elongation step at 72 °C for 7 min.
   3. For the second round PCR, use 3 µL of the first amplification product for a final volume of 40 µL containing 1.5 mM MgCl₂, 0.25 mM dNTPs, 2.5 units of DNA polymerase, and 200 nM of inner primers containing restriction enzyme sites for cloning into expression vectors (see Figure 2 and Table of Materials for primer sequences).
      NOTE: Composition of inner primers mix. For heavy chain amplification: 9 forward primers (5'AgeIVH mix) and 3 reverse primers (3'SalIJH mix). For light kappa chain amplification, 9 forward primers (5'AgeIV| mix) and 3 reverse primers (3'BsiWIJκ mix). For light lambda chain amplification, 6 forward primers (5'AgeIVl mix) and 1 reverse primer (3'XhoICl).
      NOTE: Primers were designed by Tiller et al., for amplification of all of the heavy and light chain family genes⁵.
   4. Apply the following cycling conditions: 94 °C for 4 min, followed by 40 cycles of 30 s at 94°C, 30 s at 58 °C for VH and LCκ (60 °C for LCλ), and 45 s at 72 °C with a final elongation step at 72 °C for 7 min.
5. Identify positive wells by migrating 5 μL of PCR products of vH, vLκ and vLλ on a 1.5% agarose gel (500 bp products for variable heavy chain encoding segments and a 350 bp product for variable light chain encoding segments).
6. Purify the remaining 35 µL PCR products remaining from positive wells using ultrafiltration membranes designed for purification of PCR products.
7. Elute PCR products with 60 µL of H₂O.

5. Expression Cloning

1. Prepare Inserts
   NOTE: Digest 60 µL of each purified PCR product in a final volume of 100 µL containing the appropriate restriction enzyme buffer.
   1. For vH PCR products, add 50 units of AgeI and 50 units of SalI, and incubate for 1 h at 37 °C.
   2. For vLλ PCR products, add 50 units of AgeI and 50 units of XhoI, and incubate for 1 h at 37 °C.
   3. For vLκ PCR products, add 50 units of AgeI and incubate for 1 h at 37 °C. Purify digests using 96 PCR membranes and elute with 60 µL of H₂O. Digest the 60 µL eluate in a final volume of 100 µL of BsiWI restriction enzyme buffer, add 50 units of BsiWI, and incubate for 1 h at 55 °C.
   4. Inactivate the enzymes by heating at 80 °C for 15 min.
2. Preparation of Expressing Vectors
   NOTE: vH PCR products are cloned in an expression vector (HCγ1) containing the constant heavy chain Cγ1 of IgG1. vLκ PCR products are cloned in an expression vector (LCκ) containing the constant light chain Cκ. vLλ PCR products are cloned in an expression vector (LCλ) containing the constant light chain Cλ.
   1. Perform the following digestions:
      1. Mix 1 µg of expression vector HCγ1 with 10 units of AgeI and 10 units of SalI endonucleases in a total volume of 20 µL of restriction buffer recommended by the manufacturer, and incubate for 1 h at 37 °C.
      2. Mix 1 µg of expression vector LCκ with 10 units of AgeI restriction enzyme in a total volume of 20 µL of restriction buffer recommended by the manufacturer, and incubate for 1 h at 37°C. Add 10 units of BsiWI restriction enzyme, and incubate for 1 h at 55 °C.
      3. Mix 1 µg of expression vector LCλ with 10 units of AgeI and 10 units of XhoI endonucleases in a total volume of 20 µL of restriction buffer recommended by the manufacturer, and incubate for 1 h at 37 °C
   2. Heat each digestion mixture at 65 °C for 10 min to inactivate the restriction enzymes.
   3. Perform ligation of 5 µL of digested PCR product with 2 µL (100 ng) of corresponding linearized expression vector in a total volume of 20 µL of 1x DNA ligase buffer with 1 unit of T4 DNA-Ligase by incubation overnight at 4 °C.
      NOTE: Alternatively, ligation is performed for 3 h at 4°C.
3. Cloning
   1. Electroporate 1 µL of the 20 µL ligation mixture into 45 µL of homemade electrocompetent TOP10 E. coli (Current Protocols in Molecular Biology, section 1.8.4-1.8.8).Immediately add 500 µL of 2X YT medium and incubate in a water bath at 37 °C for 30 min. Spread transformed bacteria on 2x YT ampicillin plates. Incubate overnight at 37 °C.
   2. For each transformation, screen eight colonies by PCR.
      1. Set up a reaction premix as follows on ice: 45 µL of 5x PCR Buffer, 12 µL of MgCl₂ (25 mM), 4 µL of dNTPs (from a stock containing 10 mM of each), 10 µL of forward primer (stock 10 µM) hybridizing to the vector sequence (primer Ab-vec-sense), and 10 µL of reverse primer (stock 10 µM) targeting the constant heavy or light chain region (see Table of Materials for primer sequences). Make up to a final volume of 200 µL with H₂O. Then add 4 µL of DNA polymerase enzyme (5 U/µL).
      2. Distribute 25 µL of reaction premix per 8-strip PCR tube on ice.
      3. Use a sterile toothpick to pick up individual colonies. Streak the toothpick onto a 2x TY ampicillin plate to constitute a replicate plate and then dip it into a PCR reaction tube.
      4. Perform the screening PCR under the following cycling conditions: 94 °C for 10 min, followed by 25 cycles of 30 s at 94 °C, 30 s at 55 °C, and 50 s at 72 °C.
      5. Identify positive bacteria by migrating 5 µL of the PCR products on a 1.5% agarose gel.
         NOTE: Positives colonies give a PCR product around 850 bp for the heavy chain vector and 600 bp for the light chain vector.
   3. Inoculate four positive colonies from the replicate plate into 2 mL of LB medium and incubate overnight at 37 °C with shaking at 200 rpm.
   4. Extract plasmids from liquid cultures using a plasmid purification kit, as indicated by the manufacturer.
   5. Verify correct insertion of variable domains by Sanger sequencing of the plasmids with the Ab-vec-sense primer.
      NOTE: Plasmids with the correct insert (encoding the HC and the corresponding LC) are to be cotransfected into 293A cells for secretion. The specificity of small scale-produced mAbs is assayed by ELISA. Then, large scale production is performed if applicable.

6. Production of mAbs

1. Small scale production for specificity checking
   1. The day before the transfection, seed 15,000 Human embryonic kidney 293A (HEK 293A) cells in 200 µL of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS per well in 96-well plates. Incubate overnight in a CO₂ incubator (5% CO₂) at 37 °C.
   2. Cotransfect 293A cells in DMEM medium containing 10% FBS using a linear polyethylenimine derivative as transfection reagent.
      NOTE: Perform the transfection in triplicates. The following protocol is for one well of a flat bottom 96-well plate.
      1. Dilute 0.5 µL of DNA transfection reagent into 10 µL of 150 mM NaCl. Dilute 0.125 µg of vH and 0.125 µg of vL expressing vectors into 10 µL of 150 mM NaCl. Vortex each dilution for 10 s.
      2. Add 10 µL diluted DNA transfection reagent into the 10 µL DNA solution. Vortex 15 s and incubate for 15 min at room temperature. Add the 20 µL mix drop-wise on 293A cells, and mix by gently swirling the plate.
   3. Replace the medium 16 h after transfection and culture cells for 5 days in serum-free medium.
      NOTE: Use serum-free medium to avoid serum Igs contaminating the produced antibodies.
   4. Eliminate cells and debris by centrifugation at 460 x g for 5 min.
   5. Harvest supernatants by aspiration with a multi-channel pipette and transfer them into a V-bottom 96-well plate.
   6. Coat relevant Ag overnight at 4 °C in 100 µL per well of reconstituted ELISA/ELISPOT coating buffer 1x at a final concentration of 2 µg/mL in 96-well ELISA plates. See examples in ⁹.
   7. Block wells with 10% FBS DMEM medium for 2 h at 37 °C
   8. Add 50 µL of supernatants of transfected 293A cells from step 6.1.5, and incubate for 2 h at RT.
   9. Add 100 µL of an anti-human IgG Ab conjugated to horseradish peroxidase (HRP) enzyme at 1 µg/mL and incubate for 1 h at room temperature. Add 50 µL chromogenic substrate (TMB), and incubate for 20 min.
   10. Read optical densities at 450 nm on a spectrophotometer.
       NOTE: The specific mAbs revealed by ELISA assay can be further produced at large scale and purified on protein A column presenting affinity for human IgG.
2. Large scale production and purification of specific mAbs
   1. The day before the transfection, seed 6 x 10⁶ Human embryonic kidney 293A (HEK 293A) cells into 175 cm² flasks containing 25 mL DMEM supplemented with 10% FBS. Incubate overnight in a CO₂ incubator (5% CO₂) at 37 °C.
      NOTE: Cells should be 70% confluent when transfected.
   2. Cotransfect 293A cells in DMEM medium containing 10% FBS using a linear polyethylenimine derivative as transfection reagent.
      1. Dilute 50 µL of DNA transfection reagent into 450 µL of 150 mM NaCl. Dilute 10 µg of vH and 10 µg of vL expressing vectors into 500 µL of 150 mM NaCl. Vortex 10 s each dilution.
      2. Add diluted DNA transfection reagent to the DNA solution. Vortex 15 s and incubate for 15 min at room temperature. Add the 1 mL mix drop-wise to the cells, and mix by gently swirling the flask.
   3. Replace the medium 16 h after transfection and culture cells for 5 days in serum-free medium.
   4. Harvest medium by aspirating with a 25-mL pipette.
   5. Eliminate cells and debris by centrifugation at 460 x g for 5 min.
   6. Purify the antibodies using a 1 mL column coated with protein A on a fast protein liquid chromatography (FPLC) system at 4 °C.
      1. Equilibrate the protein A sepharose column with 20 mM phosphate buffer (pH 7).
      2. Filter the cell culture medium from 6.2.5 using a 1.22 µm filter, then a 0.45 µm filter.
      3. Load the filtered medium onto the column.
      4. Wash with 20 mM phosphate buffer (pH 7) and elute with a 0.1 M citrate buffer (pH 3.5).
      5. Read optical densities at 280 nm on an absorption spectrophotometer and immediately neutralize protein-containing fractions with 1 M Tris buffer (pH 9).
   7. Dialyze purified Ab overnight at 4 °C in a cassette with a 3.5K molecular weight cutoff against PBS buffer (pH 7.4).
   8. Sterilize by 0.2 µm filtration and control purity by size-exclusion chromatography, as indicated by the manufacturer.