Single-Cell Droplet Microfluidic Screening for Antibodies Specifically Binding to Target Cells

Monoclonal antibodies are a main player in modern drug discovery. Many antibody screening formats exist, each with specific advantages and limitations. Nonetheless, it remains challenging to screen antibodies for the binding of cell-surface receptors (the most important class of all drug targets) or for the binding to target cells rather than purified proteins. Here, we present a high-throughput droplet microfluidics approach employing dual-color normalized fluorescence readout to detect antibody binding. This enables us to obtain quantitative data on target cell recognition, using as little as 33 fg of IgG per assay. Starting with an excess of hybridoma cells releasing unspecific antibodies, individual clones secreting specific binders (of target cells co-encapsulated into droplets) could be enriched 220-fold after sorting 80,000 clones in a single experiment. This opens the way for therapeutic antibody discovery, especially since the single-cell approach is in principle also applicable to primary human plasma cells

Droplet-based microfluidics in drug discovery, transcriptomics and high-throughput molecular genetics

Droplet-based microfluidics enables assays to be carried out at very high throughput (up to thousands of samples per second) and enables researchers to work with very limited material, such as primary cells, patient's biopsies or expensive reagents. An additional strength of the technology is the possibility to perform large-scale genotypic or phenotypic screens at the single-cell level. Here we critically review the latest developments in antibody screening, drug discovery and highly multiplexed genomic applications such as targeted genetic workflows, single-cell RNAseq and single-cell ChIPseq. Starting with a comprehensive introduction for non-experts, we pinpoint current limitations, analyze how they might be overcome and give an outlook on exciting future applications

Single-Cell Droplet Microfluidic Screening for Antibodies Specifically Binding to Target Cells

Summary: Monoclonal antibodies are a main player in modern drug discovery. Many antibody screening formats exist, each with specific advantages and limitations. Nonetheless, it remains challenging to screen antibodies for the binding of cell-surface receptors (the most important class of all drug targets) or for the binding to target cells rather than purified proteins. Here, we present a high-throughput droplet microfluidics approach employing dual-color normalized fluorescence readout to detect antibody binding. This enables us to obtain quantitative data on target cell recognition, using as little as 33 fg of IgG per assay. Starting with an excess of hybridoma cells releasing unspecific antibodies, individual clones secreting specific binders (of target cells co-encapsulated into droplets) could be enriched 220-fold after sorting 80,000 clones in a single experiment. This opens the way for therapeutic antibody discovery, especially since the single-cell approach is in principle also applicable to primary human plasma cells

Humanized antibody neutralizing 2009 pandemic H1N1 virus

The 2009 pandemic H1N1 S-OIV (swine origin influenza A virus) caused noticeable morbidity and mortality worldwide. In addition to vaccine and antiviral drug therapy, the use of influenza virus neutralizing monoclonal antibodies (MAbs) for treatment purposes is a viable alternative. We previously reported the isolation of a high affinity, potently neutralizing murine MAb MA2077 against 2009 pandemic H1N1 virus. We describe here the humanization of MA2077 and its expression in a mammalian cell line. Six complementarity-determining regions (CDRs) of MA2077 were grafted onto the human germline variable regions; along with six and eight back mutations in the framework of heavy and light chains, respectively, pertaining to the vernier zone and interchain packing residues to promote favorable CDR conformation and facilitate antigen binding. The full length humanized antibody, 2077Hu2, expressed in CHO-K1 cells, showed high affinity to hemagglutinin protein (K-D = 0.75 +/- 0.32 nM) and potent neutralization of pandemic H1N1 virus (IC50 = 0.17 mu g/mL), with marginally higher IC50 as compared to MA2077 (0.08 mu g/mL). In addition, 2077Hu2 also retained the epitope specificity for the ``Sa'' antigenic site on pandemic HA. To the best of our knowledge, this is the first report of a humanized neutralizing antibody against pandemic H1N1 virus

Humanized antibody neutralizing 2009 pandemic H1N1 virus

The 2009 pandemic H1N1 S-OIV (swine origin influenza A virus) caused noticeable morbidity and mortality worldwide. In addition to vaccine and antiviral drug therapy, the use of influenza virus neutralizing monoclonal antibodies (MAbs) for treatment purposes is a viable alternative. We previously reported the isolation of a high affinity, potently neutralizing murine MAb MA2077 against 2009 pandemic H1N1 virus. We describe here the humanization of MA2077 and its expression in a mammalian cell line. Six complementarity-determining regions (CDRs) of MA2077 were grafted onto the human germline variable regions; along with six and eight back mutations in the framework of heavy and light chains, respectively, pertaining to the vernier zone and interchain packing residues to promote favorable CDR conformation and facilitate antigen binding. The full length humanized antibody, 2077Hu2, expressed in CHO-K1 cells, showed high affinity to hemagglutinin protein (KD = 0.75 ± 0.32 nM) and potent neutralization of pandemic H1N1 virus (IC50 = 0.17 μg/mL), with marginally higher IC50 as compared to MA2077 (0.08 μg/mL). In addition, 2077Hu2 also retained the epitope specificity for the “Sa” antigenic site on pandemic HA. To the best of our knowledge, this is the first report of a humanized neutralizing antibody against pandemic H1N1 virus

Isolation of a High Affinity Neutralizing Monoclonal Antibody against 2009 Pandemic H1N1 Virus That Binds at the ‘Sa’ Antigenic Site

<div><p>Influenza virus evades host immunity through antigenic drift and shift, and continues to circulate in the human population causing periodic outbreaks including the recent 2009 pandemic. A large segment of the population was potentially susceptible to this novel strain of virus. Historically, monoclonal antibodies (MAbs) have been fundamental tools for diagnosis and epitope mapping of influenza viruses and their importance as an alternate treatment option is also being realized. The current study describes isolation of a high affinity (<em>K</em>_D = 2.1±0.4 pM) murine MAb, MA2077 that binds specifically to the hemagglutinin (HA) surface glycoprotein of the pandemic virus. The antibody neutralized the 2009 pandemic H1N1 virus in an <em>in vitro</em> microneutralization assay (IC₅₀ = 0.08 µg/ml). MA2077 also showed hemagglutination inhibition activity (HI titre of 0.50 µg/ml) against the pandemic virus. In a competition ELISA, MA2077 competed with the binding site of the human MAb, 2D1 (isolated from a survivor of the 1918 Spanish flu pandemic) on pandemic H1N1 HA. Epitope mapping studies using yeast cell-surface display of a stable HA1 fragment, wherein ‘Sa’ and ‘Sb’ sites were independently mutated, localized the binding site of MA2077 within the ‘Sa’ antigenic site. These studies will facilitate our understanding of antigen antibody interaction in the context of neutralization of the pandemic influenza virus.</p> </div

Expression of designed HA1 constructs on yeast cell surface.

<p>(A–C) Histograms depicting the fluorescence signal of Alexa Fluor-488 labeled antibody bound to the C-terminal c-Myc tag of the displayed proteins. The Alexa Fluor-488 signal indicates the cell-surface expression of HA1 fragments. (D) The designed fragment, H1pHA9, is well folded and bound the conformationally sensitive MAb 2D1 which was detected with PE-labeled antibody.</p

<i>In vitro</i> neutralization and HI activity of MA2077.

a<p>Specific activity of MAb is the minimum concentration of the antibody required to neutralize 10% of 100 TCID₅₀ dose of the virus in an <i>in vitro</i> microneutralization assay or to inhibit 4 HA units of the virus in HI assay using 1% guinea pig RBCs.</p><p>>25 indicates that inhibitory activity was not detected at any concentration upto 25 µg/ml.</p><p>NA- Not applicable.</p

Design of H1pHA9(Sa) and H1pHA9(Sb) constructs.

<p>The ‘Sa’ and ‘Sb’ antigenic sites were independently mutated in our designed fragment, H1pHA9. (A) ‘Sa’ antigenic site residues are colored cyan. The mutations introduced in H1pHA9(Sa) to disrupt this epitope are labeled (colored blue). (B) ‘Sb’ antigenic site residues are colored light pink. The mutations introduced in H1pHA9(Sb) are labeled (colored pink). (A, B) Mutations introduced in H1pHA9 to remove exposed hydrophobic patches are in red. The receptor binding site residues are in yellow. The figures were generated in PyMOL (The PyMOL Molecular Graphics System, version 1.2r2, DeLano Scientific, LLC).</p

Design of H1pHA9 fragment.

<p>An HA1 fragment, H1pHA9, defined by stable breakpoints was designed <i>in silico</i>. (A) Shown in stereoview is the cartoon representation of H1N1 A/California/04/2009 HA trimer (PDB ID: 3LZG). Residues 65–286 comprising H1pHA9 (colored grey) have been mapped onto a monomer (orange) of the crystal structure of H1N1 A/California/04/2009 HA. The rest of the molecule is colored green. The mutations introduced in H1pHA9 are colored red. The receptor binding site residues are colored yellow. (B) The mutated residues (red) in H1pHA9 do not contribute to any of the antigenic sites or the receptor binding site (yellow) on HA. The figures were generated in PyMOL (The PyMOL Molecular Graphics System, version 1.2r2, DeLano Scientific, LLC).</p