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Generation of Recombinant Monoclonal Antibodies from Immunised Mice and Rabbits via Flow Cytometry and Sorting of Antigen-Specific IgG+ Memory B Cells.

Single B cell screening strategies, which avoid both hybridoma fusion and combinatorial display, have emerged as important technologies for efficiently sampling the natural antibody repertoire of immunized animals and humans. Having access to a range of methods to interrogate different B cell subsets provides an attractive option to ensure large and diverse panels of high quality antibody are produced. The generation of multiple antibodies and having the ability to find rare B cell clones producing IgG with unique and desirable characteristics facilitates the identification of fit-for-purpose molecules that can be developed into therapeutic agents or research reagents. Here, we describe a multi-parameter flow cytometry single-cell sorting technique for the generation of antigen-specific recombinant monoclonal antibodies from single IgG+ memory B cells. Both mouse splenocytes and rabbit PBMC from immunised animals were used as a source of B cells. Reagents staining both B cells and other unwanted cell types enabled efficient identification of class-switched IgG+ memory B cells. Concurrent staining with antigen labelled separately with two spectrally-distinct fluorophores enabled antigen-specific B cells to be identified, i.e. those which bind to both antigen conjugates (double-positive). These cells were then typically sorted at one cell per well using FACS directly into a 96-well plate containing reverse transcriptase reaction mix. Following production of cDNA, PCR was performed to amplify cognate heavy and light chain variable region genes and generate transcriptionally-active PCR (TAP) fragments. These linear expression cassettes were then used directly in a mammalian cell transfection to generate recombinant antibody for further testing. We were able to successfully generate antigen-specific recombinant antibodies from both the rabbit and mouse IgG+ memory B cell subset within one week. This included the generation of an anti-TNFR2 blocking antibody from mice with an affinity of 90 pM

Schematic representation of the single-B cell sorting protocol used for antibody discovery from immunised mice.

<p>The following steps were undertaken: 1. Mice were immunised and a splenocyte suspension prepared. 2. B cells from the mouse splenocyte preparation were enriched using anti-mouse CD45R microbeads (Miltenyi Biotech) and LS MACS columns (Miltenyi Biotech) according to manufacturers’ instructions. 3. Following enrichment, cells were stained with the following antibodies: rat anti-mouse IgG brilliant violet 421 (BD biosciences), rat anti-mouse IgM PE-Cy7 (Bio legend), rat anti-mouse IgD APC-Cy7 (Bio legend), rat anti-mouse CD19 AF700 (BD biosciences) (2 μg per 108 Cells), and rat anti-mouse CD4, CD8, GR1 and F4/80 FITC (BD biosciences)(dump channel). Human TNFR2 extracellular domain was labelled with PE and APC using Lightning Link PE and APC labelling kits (Innova Bioscience) and added to the cell suspension. 4. FACS was performed on a BD FACS ARIA III with single human TNFR2-specific IgG⁺ B cells being deposited into the well of a 96-well PCR plate. 5. cDNA from single B cells was prepared using Superscript III reverse transcriptase (Invitrogen) primed with oligo (dT). Antibody variable-region genes were then recovered via two rounds of PCR followed by a third round to generate transcriptionally-active PCR (TAP) products in a manner similar to that described in Clargo <i>et al</i>.⁹ employing an Aviso Onyx liquid handling robot to facilitate set-up. 6. Heavy and light chain TAP fragments were transiently co-transfected into Expi293 cells using ExpiFectamine (Life Technologies). After 7 days expression, supernatants were harvested for further characterisation.</p

Gating strategy for the identification of antigen-specific mouse memory B cells from IL-25 immunised mice.

<p>Following cell enrichment using CD45R microbeads (Miltenyi Biotech), cells were analysed in a BD FACS ARIA III. A gate was drawn around the lymphocyte population (gated population represented 47.6% of events) (A). FSC-W and FSC-A were then used to eliminate doublets (gated population represented 99% of events) (B). T cells, macrophages, neutrophils and 7AAD⁺ dead cells were eliminated in the “dump channel” (gated population represented 95.8% of events) (C). CD19⁺ B cells were identified (gated population represented 99% of events) (D). IgG⁺/IgM^- B cells were then gated on (gated population represented 1.49% of events) (E). To further eliminate naïve B cells, IgD staining allowed gating for IgG⁺/IgD^- cells, (gated population represented 91% of events) (F). Finally, dual-colour antigen staining allowed a gate (P1) to be drawn around the double-positive population (gated population P1 represented 0.283% of events) (G). Single cells from gate P1 were sorted into a 96-well plate for single-cell RT-PCR.</p

Summary of recombinant IgG expression and binding recoveries.

<p>Summary of recombinant IgG expression and binding recoveries.</p

Gating strategy for identification of antigen-specific rabbit memory B cells.

<p>Cells were analysed in a BD FACS ARIA III. A gate was drawn around the lymphocyte population (gated population represented 59.8% of events) (A). FSC-W and FSC-A were then used to eliminate doublets (gated population represented 95.7% of events) (B). 7AAD⁺ dead cells and T cells were eliminated in the “dump channel” (gated population represented 97.1% of events) (C). IgG⁺/ IgM^- B cells were identified and gated on (gated population represented 2.67% of events) (D). Finally, a gate (P1) was drawn around the double-positive mWISP-1 antigen-specific population (gated population represented 0.262% of events) (E). Cells from gate P1 were sorted into a 96-well PCR plate at either one or three cells per well for subsequent RT-PCR.</p

Schematic representation of the single-B cell sorting protocol used for antibody discovery from immunised mice.

<p>The following steps were undertaken: 1. Mice were immunised and a splenocyte suspension prepared. 2. B cells from the mouse splenocyte preparation were enriched using anti-mouse CD45R microbeads (Miltenyi Biotech) and LS MACS columns (Miltenyi Biotech) according to manufacturers’ instructions. 3. Following enrichment, cells were stained with the following antibodies: rat anti-mouse IgG brilliant violet 421 (BD biosciences), rat anti-mouse IgM PE-Cy7 (Bio legend), rat anti-mouse IgD APC-Cy7 (Bio legend), rat anti-mouse CD19 AF700 (BD biosciences) (2 μg per 108 Cells), and rat anti-mouse CD4, CD8, GR1 and F4/80 FITC (BD biosciences)(dump channel). Human TNFR2 extracellular domain was labelled with PE and APC using Lightning Link PE and APC labelling kits (Innova Bioscience) and added to the cell suspension. 4. FACS was performed on a BD FACS ARIA III with single human TNFR2-specific IgG⁺ B cells being deposited into the well of a 96-well PCR plate. 5. cDNA from single B cells was prepared using Superscript III reverse transcriptase (Invitrogen) primed with oligo (dT). Antibody variable-region genes were then recovered via two rounds of PCR followed by a third round to generate transcriptionally-active PCR (TAP) products in a manner similar to that described in Clargo <i>et al</i>.⁹ employing an Aviso Onyx liquid handling robot to facilitate set-up. 6. Heavy and light chain TAP fragments were transiently co-transfected into Expi293 cells using ExpiFectamine (Life Technologies). After 7 days expression, supernatants were harvested for further characterisation.</p

Gating strategy for identification of antigen-specific mouse memory B cells from TNFR2 immunised mice.

<p>Following cell enrichment using CD45R microbeads (Miltenyi Biotech), cells were analysed in a BD FACS ARIA III. A gate was drawn around the lymphocyte population (gated population represented 34.9% of events) (A). FSC-W and FSC-A were then used to eliminate doublets (gated population represented 95.1% of events) (B). T cells, macrophages, neutrophils and 7AAD⁺ dead cells were eliminated in the “dump channel” (gated population represented 95.4% of events) (C). CD19⁺ B cells were identified (gated population represented 97.2% of events) (D). IgG⁺/IgM^- B cells were then gated on (gated population represented 0.899% of events) (E). To further eliminate naïve B cells, IgD staining allowed gating for IgG⁺/IgD^- cells (gated population (gate P1) represented 96.3% of events) (F). In order to demonstrate the importance of the antigen staining step, we sorted single cells from the total IgG⁺/IgD^- cell population (gate P1). Finally, dual-colour antigen staining allowed a gate (P2) to be drawn around the double-positive antigen-specific population (gated population P2 represented 0.95%of events) (G). Single cells from gate P2 were sorted into a 96-well plate for single-cell RT-PCR.</p

Recombinant IgG concentration range produced from IL-25 B cell sorting experiment.

<p>Heavy and light chain TAP fragments produced from single-sorted B cells were transiently co-transfected into Expi293 using ExpiFectamine (Life Technologies). After 7 days expression, supernatants were harvested and the concentration of mouse IgG measured using an sandwich ELISA with a purified mouse IgG standard. Data is shown for all wells that produced measurable antibody production. Green bars indicate those that were determined to bind IL-25 and red and black-check bars represent those which did not show binding to IL-25.</p