Generation of a single-cell B cell atlas of antibody repertoires and transcriptomes to identify signatures associated with antigen specificity.

Although new genomics-based pipelines have potential to augment antibody discovery, these methods remain in their infancy due to an incomplete understanding of the selection process that governs B cell clonal selection, expansion, and antigen specificity. Furthermore, it remains unknown how factors such as aging and reduction of tolerance influence B cell selection. Here we perform single-cell sequencing of antibody repertoires and transcriptomes of murine B cells following immunizations with a model therapeutic antigen target. We determine the relationship between antibody repertoires, gene expression signatures, and antigen specificity across 100,000 B cells. Recombinant expression and characterization of 227 monoclonal antibodies revealed the existence of clonally expanded and class-switched antigen-specific B cells that were more frequent in young mice. Although integrating multiple repertoire features such as germline gene usage and transcriptional signatures failed to distinguish antigen-specific from nonspecific B cells, other features such as immunoglobulin G (IgG) subtype and sequence composition correlated with antigen specificity.

Phenotypic determinism and stochasticity in antibody repertoires of clonally expanded plasma cells.

The capacity of humoral B cell-mediated immunity to effectively respond to and protect against pathogenic infections is largely driven by the presence of a diverse repertoire of polyclonal antibodies in the serum, which are produced by plasma cells (PCs). Recent studies have started to reveal the balance between deterministic mechanisms and stochasticity of antibody repertoires on a genotypic level (i.e., clonal diversity, somatic hypermutation, and germline gene usage). However, it remains unclear if clonal selection and expansion of PCs follow any deterministic rules or are stochastic with regards to phenotypic antibody properties (i.e., antigen-binding, affinity, and epitope specificity). Here, we report on the in-depth genotypic and phenotypic characterization of clonally expanded PC antibody repertoires following protein immunization. We find that clonal expansion drives antigen specificity of the most expanded clones (top ∼10), whereas among the rest of the clonal repertoire antigen specificity is stochastic. Furthermore, we report both on a polyclonal repertoire and clonal lineage level that antibody-antigen binding affinity does not correlate with clonal expansion or somatic hypermutation. Last, we provide evidence for convergence toward targeting dominant epitopes despite clonal sequence diversity among the most expanded clones. Our results highlight the extent to which clonal expansion can be ascribed to antigen binding, affinity, and epitope specificity, and they have implications for the assessment of effective vaccines.

Cyto-Mine: An Integrated, Picodroplet System for High-Throughput Single-Cell Analysis, Sorting, Dispensing, and Monoclonality Assurance.

The primary goal of bioprocess cell line development is to obtain high product yields from robustly growing and well-defined clonal cell lines in timelines measured in weeks rather than months. Likewise, high-throughput screening of B cells and hybridomas is required for most cell line engineering workflows. A substantial bottleneck in these processes is detecting and isolating rare clonal cells with the required characteristics. Traditionally, this was achieved by the resource-intensive method of limiting dilution cloning, and more recently aided by semiautomated technologies such as cell sorting (e.g., fluorescence-activated cell sorting) and colony picking. In this paper we report on our novel Cyto-Mine Single Cell Analysis and Monoclonality Assurance System, which overcomes the limitations of current technologies by screening hundreds of thousands of individual cells for secreted target proteins, and then isolating and dispensing the highest producers into microtiter plate wells (MTP). The Cyto-Mine system performs this workflow using a fully integrated, microfluidic Cyto-Cartridge. Critically, all reagents and Cyto-Cartridges used are animal component-free (ACF) and sterile, thus allowing fast, robust, and safe isolation of desired cells.

Rapid isolation of antigen-specific B-cells using droplet microfluidics.

Monoclonal antibodies are powerful tools for scientific research and are the basis of numerous therapeutics. However, traditional approaches to generate monoclonal antibodies against a desired target, such as hybridoma-based techniques and display library methods, are laborious and suffer from fusion inefficiency and display bias, respectively. Here we present a platform, featuring droplet microfluidics and a bead-based binding assay, to rapidly identify and verify antigen-binding antibody sequences from primary cells. We used a defined mixture of hybridoma cells to characterize the system, sorting droplets at up to 100 Hz and isolating desired hybridoma cells, comprising 0.1% of the input, with a false positive rate of less than 1%. We then applied the system to once-frozen primary B-cells to isolate rare cells secreting target-binding antibody. We performed RT-PCR on individual sorted cells to recover the correctly paired heavy- and light-chain antibody sequences, and we used rapid cell-free protein synthesis to generate single-chain variable fragment-format (scFv) antibodies from fourteen of the sorted cells. Twelve of these showed antigen-specific binding by ELISA. Our platform facilitates screening animal B-cell repertoires within days at low cost, increasing both rate and range of discovering antigen-specific antibodies from living organisms. Further, these techniques can be adapted to isolate cells based on virtually any secreted product.

Epitope determines efficacy of therapeutic anti-Tau antibodies in a functional assay with human Alzheimer Tau.

In Alzheimer's disease (AD) and other tauopathies, the cytosolic protein Tau misfolds and forms intracellular aggregates which accumulate within the brain leading to neurodegeneration. Clinical progression is tightly linked to the progressive spread of Tau pathology throughout the brain, and several lines of evidence suggest that Tau aggregates or "seeds" may propagate pathology by spreading from cell to cell in a "prion like" manner. Accordingly, blocking the spread of extracellular seeds with an antibody could be a viable therapeutic approach. However, as the structure of Tau seeds is unknown, it is only possible to rationally design therapeutic Tau antibodies by making a priori assumptions. To avoid this, we developed a robust and quantitative cell based assay and employed an unbiased screening approach to identify the antibody with the highest activity against human Tau seeds. The selected antibody (D), directed to the mid-region of Tau (amino acids 235-250), potently blocked the seeding of human AD Tau and was also fully efficacious against seeds from progressive supranuclear palsy. When we compared this antibody with previously described reference antibodies, we were surprised to find that none of these antibodies showed comparable efficacy against human pathological seeds. Our data highlight the difficulty of predicting antibody accessible epitopes on pathological Tau seeds and question the potential efficacy of some of the Tau antibodies that are currently in clinical development.

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.

A fully automated primary screening system for the discovery of therapeutic antibodies directly from B cells.

For a therapeutic antibody to succeed, it must meet a range of potency, stability, and specificity criteria. Many of these characteristics are conferred by the amino acid sequence of the heavy and light chain variable regions and, for this reason, can be screened for during antibody selection. However, it is important to consider that antibodies satisfying all these criteria may be of low frequency in an immunized animal; for this reason, it is essential to have a mechanism that allows for efficient sampling of the immune repertoire. UCB's core antibody discovery platform combines high-throughput B cell culture screening and the identification and isolation of single, antigen-specific IgG-secreting B cells through a proprietary technique called the "fluorescent foci" method. Using state-of-the-art automation to facilitate primary screening, extremely efficient interrogation of the natural antibody repertoire is made possible; more than 1 billion immune B cells can now be screened to provide a useful starting point from which to identify the rare therapeutic antibody. This article will describe the design, construction, and commissioning of a bespoke automated screening platform and two examples of how it was used to screen for antibodies against two targets.

The rapid generation of recombinant functional monoclonal antibodies from individual, antigen-specific bone marrow-derived plasma cells isolated using a novel fluorescence-based method.

Single B cell technologies, which avoid traditional hybridoma fusion and combinatorial display, provide a means to interrogate the naturally-selected antibody repertoire of immunized animals. Many methods enable the sampling of memory B cell subsets, but few allow for the direct interrogation of the plasma cell repertoire, i.e., the subset of B cells responsible for producing immunoglobulin in serum. Here, we describe the use of a robust and simple fluorescence-based technique, called the fluorescent foci method, for the identification and isolation of antigen-specific IgG-secreting cells, such as plasma cells, from heterogeneous bone marrow preparations. Following micromanipulation of single cells, cognate pairs of heavy and light chain variable region genes were recovered by reverse transcription (RT)-polymerase chain reaction (PCR). During the PCR, variable regions were combined with a promoter fragment and a relevant constant region fragment to produce two separate transcriptionally-active PCR (TAP) fragments that were directly co-transfected into a HEK-293F cell line for recombinant antibody expression. The technique was successfully applied to the generation of a diverse panel of high-affinity, functional recombinant antibodies to human tumor necrosis factor (TNF) receptor 2 and TNF derived from the bone marrow of immunized rabbits and rats, respectively. Progression from a bone marrow sample to a panel of functional recombinant antibodies was possible within a 2-week timeframe.