Join with all of our speakers for a roundtable discussion!

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.

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.

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.

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.

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.

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.

Poster Abstract