Poster Abstract

Antibody affinity is a critical metric for antibody therapeutic development and is often only assessed after initial antibody discovery hit generation. However, due to the cost and throughput of small-scale antibody production, antibody affinity characterization is only reserved for a few lead candidates. Here, we demonstrate a high-throughput relative affinity ranking of recombinant antigen-specific antibodies during the antibody discovery workflow on Bruker’s Beacon optofluidic system.

Background: T cells are heterogeneous in both phenotype and function and have variable responses to stimulation. Existing methods to assess T cell function are typically applied individually to separate cell populations such that relationships between functional parameters such as cytotoxicity and gene expression can only be inferred. We previously demonstrated the ability to rapidly evaluate cytokine secretion and cytotoxicity at single-cell resolution for thousands of cells in parallel. Transcriptomic analysis was performed on T cells that were activated with antigen-presenting beads and the results were correlated to T cell polyfunctionality. When antigen-presenting cells are present in the co-culture, downstream processing has been limited to T cell-specific readouts due to contaminating RNA transcripts. Here, we describe a high-throughput, automated workflow to isolate single cells of interest sequentially from a transient co-culture for downstream single-cell analysis. This workflow was demonstrated on dividing T cells for lineage tracking and may be applied toward assays requiring co-culture of multiple cell types.

Methods: T cells from a healthy donor were activated with CD3/CD28 beads and labeled with CellTrace Far Red. Single T cells were isolated into NanoPen® chambers on OptoSelect® chips using optoelectronic positioning (OEP®). Single anti-CD3/CD28 activation beads were added to each NanoPen chamber and the cells were cultured with timelapse fluorescence imaging in 30-minute intervals to identify cell division events. Once sufficient cell division was observed, 192 T cells were isolated sequentially from 96 T cell pairs and recovered for downstream processing. Fluorescent images acquired during the cell recovery were used to map cells exported from specific NanoPen chambers to their respective well plate locations.

Results: Over 3,000 single T cells were isolated for overnight culture. Detectable cell division was observed in 26% of pens after 7 hours of culture. T cells were sequentially isolated from 94 of the 96 T cell pairs attempted and the fluorescence intensity of each cell was recorded.

Conclusions: The Beacon® Optofluidic System enables multi-dimensional characterization of T cell potency at single-cell resolution. Here, we expand on these capabilities and demonstrate the ability to isolate individual T cells sequentially from multicell cultures for downstream single-cell processing. Fluorescent images captured during single-cell isolation allow for tracing of specific cells or cell types, such as T cells and tumor cells in a co-culture, to sequencing results. This capability can be applied toward correlation of on-chip phenotypic assay data, including cytokine secretion, cytotoxicity, or surface expression to single-cell RNA sequencing that was previously not feasible.

Traditional methodologies to quantify growth and production rates for antibody-expressing CHO clones involve repeatedly sampling aliquots and supernatants from well plates that have been seeded with single cells. The number of clones studied is often limited by cloning efficiencies (typically 5-50%) and the inability to handle large numbers of well plates. Measurement cadence is limited by the growth rates of cells and the number of cells required to perform each assay. These factors limit the practical throughput of screening clones to 100s over the course of 2-4 weeks. Furthermore, measurements of clone behavior at small scales often correlate poorly with larger fermenter reactors due to significantly different culture environments.

Bruker Cellular Analysis has developed a platform that uses disposable nanofluidic chips to screen 5000 clones in individual nanoscale bioreactors automatically within 5 days. Individual cells are placed in separate chambers (NanoPens) using light-induced dielectrophoresis. Clones are continuously perfused in tightly-controlled reactor-relevant microenvironments leading to 60-80%cloning efficiency. Clones are imaged and analyzed with a deep learning algorithm to provide very high-fidelity morphology and growth rate characteristics. Antibody production is measured with a quantitative secretion assay using fluorescently-tagged small molecules, independently measuring all clones simultaneously. Selected clones can be recovered from the chip for scale up.

In collaboration with Daiichi Sankyo (DS), two applications were explored. First, a head-to-head comparison of DS’s current clone screening methodology and the Beacon workflow was performed on a transfected pool. Second, previously characterized clones were sub-cloned on the Beacon and the behavior of each clone was measured over the course of 8 weeks. Analysis demonstrated the ability to measure and predict the stability of each clonal cell line.

The revolution in life science research aims to understand how single cells work, which is crucial for developing new therapies. Traditional methods like FACS and scRNASeq give only a partial view of cells. Bruker's Beacon® Optofluidic System brings a fresh approach, offering a complete picture of single cell function. The Beacon enables serial multi-modal analysis and real time imaging on the same live single cell, with 10,000s of cells simultaneously. It uses two advanced technologies: 1) Light driven Opto-Electro Positioning (OEP) system to precisely control single cells and beads movement, allowing customized experiments without harming the cells. 2) The microfluidic chip with NanoPen® chambers for continuous media and assay reagent flow, letting researchers study the same cell over time with a high-resolution microscope. The Beacon also lets researchers move selected cells from the chip for further study, linking on-chip phenotype data with off-chip sequencing or for scaling the cells of interest. Users can leverage preset workflows tailored for various applications such as CAR/TCR discovery, T-cell Profiling, antibody discovery, and cell line development, or customize experiments according to their needs. In summary, Bruker's Beacon® Optofluidic platform marks a significant advancement in single cell functional biology, poised to drive impactful research outcomes by providing researchers with unparalleled insights into cellular dynamics. This innovation has the potential to lead to tangible improvements in Antibody and Cell Therapy development, ultimately driving the next era of functional cell biology to advance human health.

Cancer immunotherapy approaches such as adoptive transfer of chimeric antigen receptor (CAR) T cells or tumor-infiltrating lymphocytes have yielded promising results in hematological malignancies. Despite inducing deep responses in multiple myeloma, both antigen escape and limited persistence lead to CAR-T cell therapy resistance. Since multiple myeloma remains an incurable malignancy, novel therapeutic approaches are urgently needed. Adoptive transfer of T cells expressing an engineered T cell receptor (TCR-T cells) represent a promising therapeutic alternative. In contrast to CAR-T cells, TCR-T cells can additionally target intracellular antigens, thereby expanding the range of potential immunotherapeutic targets. Furthermore, TCR-T cell therapy may lead to prolonged persistence in vivo and could mediate sustained anti-tumor effects.

In our project, we identified TCRs specifically targeting autologous myeloma cells. Myeloma-reactive T cells were identified applying nanofluidics and opto-electropositioning using the Bruker Cellular Analysis Lightning® platform. This enabled simultaneous functional analysis of up to 1500 individual T cell/target cell interactions on a chip. Myeloma-reactive T cells were discovered based on the secretion of cytokines (IFNγ, IL-2, TNFα) and surface expression of 4-1BB (CD137). Among 15 myeloma patients tested, reactive T cells demonstrating various cytokine secretion patterns and 4-1BB expression were detected (on average 1.2% of T cells screened). Individual myeloma-reactive T cells were isolated and their respective TCR was sequenced. Additionally, single-cell RNA sequencing was conducted for all 15 patients. By mapping TCR sequences of exported T cells to the single-cell RNA sequencing data, a gene expression signature of myeloma-reactive T cells was established. Genomic TCR sequences were in vitro transcribed and electroporated into autologous CD8+ T cells, followed by co-culture with myeloma cells or PBMCs as negative control. Myeloma-reactivity of transgenic T cells was determined for three patients (n = 11 TCRs) by flow cytometric measurement of T cell activation markers CD69 and 4-1BB. Through immuno-precipitation of MHC I molecules and mass spectrometry of the eluted peptides, we identified T cell responses against both shared and private cancer antigens for 12 patients.

To summarize, we established a pipeline for the identification and functional validation of myeloma-reactive T cells. Together with the evidence of expressed shared and private myeloma derived antigens, we envision this approach to facilitate patient-individualized T cell therapy for multiple myeloma patients.

In both the research and clinical setting, being able to identify and collect biologically meaningful data related to productive antitumor responses is of paramount importance when trying to evaluate the efficacy of a particular cell-based cancer immunotherapy. Historically, this has been a major bottleneck for investigators as many of the available technologies provide readouts of limited utility due to constraints surrounding experimental throughput, end-point readouts where a single measurement is performed after some arbitrary fixed amount time, and the inability to link multi-modal data back to single cells of interest. In his talk, Dr. Anthony E. Zamora will highlight current research being performed in his lab that highlight how the Beacon platform and real-time biological readouts can be leveraged to help unravel the biological complexities that exist across many of today’s leading cell-based therapies for cancer and how multi-modal data can be used to gain insight into the biological parameters that are associated with clinical efficacy.

Despite demonstrated efficiency in antibody generation, classical immunization strategies and subsequent hybridoma generation often face strong limitations when it comes to speed and poorly immunogenic membrane proteins with short extracellular domains. Indeed, even if a few antibodies can be obtained with repeated campaigns, only limited diversity and molecular characteristics are sometimes achieved, resulting in difficulties in selecting good candidates for pharmaceutical developments. Innovative approaches combining RNA immunization and single cell screening provide unique opportunities to dramatically speed up antibody discovery against such challenging targets. In the presentation, rapide obtention of large collections of antibodies with both molecular and function diversity on ‘classical’ targets as well as against a difficult GPCR and ion channel will be examplified using these strategies.

As one of the earliest partners and the first one in Asia-Pacific region of Berkeley Lights (now Bruker Cellular Analysis), Nona Biosciences has built a state-of-the-art single B cell cloning antibody discovery platform utilizing the Beacon system in combination with our proprietary fully human Harbour Mice® technology. In this talk, besides the usual single B cell cloning workflow, we’ll also share with you case studies on the exploration of 1) single B cell cloning + NGS to expand the throughput, 2) Beacon in-chip assay with overnight cultured or frozen plasma cells, the screening efficiency and diversity coverage, 3) some tips for assay optimization.

Cell line development with high quality and regulatory-compliant materials is very important for biopharmaceutical production. The traditional process is time consuming and labor intensive. The Beacon Optofluidic system from Bruker Cellular Analysis accelerates this process by automatically screening thousands of clones for antibody production. However, creating a cell line that expresses bispecific antibodies (BiAb) presents additional challenges due to the heterogeneity of the resulting products, including heterodimers, halfmers, and homodimers from each single arm.  In order to select the best clone, we need to be able to accurately measure the percentage of heterodimers in bispecific antibodies.

To address this challenge, we collaborated with Bruker Cellular Analysis to develop a beacon on-chip binding assay. First, we labeled one of the ligands with Alexa-594, this antigen binds specifically to single arm-A of the bispecific antibody. Next, we performed an on-chip binding assay using the labeled ligand A and SpotLight Hu Fc assay, which utilizes a specialized reagent designed to detect the antibody’s Fc part. Analysis of the fluorescence intensity of the dyes enabled us to calculate the stoichiometric ratio of arm-A to Fc.

To validate our assay, we compared the ratios determined by the on-chip binding assay and the actual ratios obtained from previous bulk pools, mini pools and clones. The results revealed a strong correlation affirming the accuracy and reliability of the on-chip assay. Then we selected clones from a bulk pool with different arm-A to Fc ratio and SpotLight Hu Fc score and analyzed fed-batch titer and product quality. The results indicated that the on-chip binding assay not only streamlined the cell line development process but also significantly improved the efficiency.

Join with all of our speakers for a roundtable discussion!