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ABSTRACT

CD28 is a costimulatory receptor for T-cell activation, and CD28 binding antibodies are in clinical development to block or promote T-cell activation[1,2] . CD28-specific binding domains are also used in bi- or tri-specific antibody formats to generate T-cell engaging antibodies [3] . We set out to generate CD28-specific mAbs from rabbits using a single B-cell cloning workflow on the Beacon system.

The Beacon system enables rapid antibody discovery using a function-first approach. Antibody-secreting cells (ASCs) are isolated from immunized animals and placed individually into nanowells (pens) of a Beacon chip. Due to the small size of the pens, the secreted antibody concentration reaches µg/ml levels within minutes, and the functional properties of the antibodies can be tested in sequential or multiplexed bead-based assays with a fluorescent readout. Typical assay duration is 30-60 minutes. Rabbits were immunized with CHOK1 cells stably transfected to carry human CD28 (hCD28) on their surface. Serum samples showed clear, yet low specific titers for hCD28.

At the end of the immunization schedule, memory B-cells were isolated from blood using Ficoll separation and magnetic sorting. B-cells were cultured in an activation media, and cells were analyzed after 4 and 5 days of activation. During the activation period, the average cell size increased and cells proliferated roughly 15-fold until day 5, with cell numbers unchanged after that. Flow cytometry showed a reduction of surface IgG and a clear increase of intracellular IgG during activation, consistent with a shift from Bcells to antibody secreting cells. On day 4 and 5, a total of 32,716 activated B-cells were screened on Beacon, first for overall IgG secretion and then for IgG specific for hCD28. 14,889 cells secreted IgG (76%), and 108 cells secreted IgG specific to hCD28 (0.43% of IgG secretors).

cDNA from identified hits was individually exported from the chip and submitted for VH/VL sequencing. Antibody discovery on the Beacon enabled rapid screening of a large number of ASCs and identification of antigen-specific antibodies within 4 days of obtaining PBMCs from immunized rabbits. cDNA generation and export happened on the day of the Beacon run, and cDNA amplification added another day to the workflow. The method was robust and substantially reduced the hands-on time for rabbit antibody discovery.

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ABSTRACT

PD-L1 is a key inhibitor of T cell activation that is often over-expressed in cancer to escape immune surveillance and promote tumor progression. Blocking antibodies against PD-L1 or its receptor, PD-1, have shown significant clinical benefit in some patients with PD-L1 expressing tumors. Hence, there is great interest in generating therapeutic antibodies against these targets to counteract the immune suppression mechanism that tumors rely on for survival.

Most of the anti-PD-L1 therapies in the clinic have been generated by standard hybridoma technology but it remains to be determined whether superior antibodies with greater diversity, affinity, and/or functional activity could be generated using single B cell cloning. Using the Beacon® platform (Berkeley Lights, Inc., (BLI)), we have screened tens of thousands of primary single plasma B cells to identify unique antibodies against PD-L1. We have immunized Balb/c mice with recombinant Fc fusion of human PD-L1 extracellular domain (ECD) and isolated CD138+ plasma B cells from bone marrow and spleen from an 8 week immunization.

Plasma B cells from both spleen and bone marrow were penned as single cells onto OptoSelectTM chips for analysis. A series of assays were performed in tandem including bead-based binding/blocking assays, cell-based binding to CHOK1 cells engineered to overexpress human PD-L1, and cell-based blocking assays using PD-1. This series of assays on the Beacon enabled identification of roughly 300 antibodies binding to PD-L1, some of which blocked binding of PD-1 to PD-L1 on cells. Interestingly, a majority of PD-L1 specific antibodies were identified from plasma B cells isolated from bone marrow even though the yield of plasma B cells from bone marrow was less than 20% of what was obtained from the spleen. Single plasma B cells were exported by the Beacon platform for antibody sequence recovery.

After reverse-transcription, amplification, and single plasma B cell sequencing by NGS, we evaluated the sequence diversity of anti-PD-L1 antibody hits. Taken together, our results from plasma B cell cloning on the Beacon show that plasma B cells secreting functional antibody candidates can be identified within one week compared to 1-2 months for the standard hybridoma campaign, thus substantially accelerating the antibody discovery process.

In addition, this technology could enable rapid comparison of immunization strategies, mouse strains, and lymphoid organs from which to isolate B cells to maximize recovery of target specific functional antibodies representing the diversity of the immune repertoire.

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ABSTRACT

The monoclonal antibody (mAb) market keeps growing strongly with a CAGR of >14% and is expected to reach >$450 billion in 2028. One remaining challenge is the efficient discovery of mAbs with the desired properties, like high functional activity, high affinity, low immunogenicity, long halflife, and cross-reactivity to target homologues in other species to enable animal models. Conventional antibody discovery programs are typically based on the generation of many potential hits followed by tedious downselection of a few good leads.

Novel technologies like Berkeley Lights’ (BLI) single plasma cell cloning platform, Beacon®, promise to drastically simplify and shorten antibody discovery workflows by identifying a much reduced number of highly functional hits within a few hours of sacrificing the animal. This is achieved by placing thousands of individual plasma cells from the immunized animal into nano-wells called pens, and testing multiple properties of the secreted antibodies in a short time frame. In order to compare the functionality of the Beacon platform with a hybridoma approach we generated monoclonal antibodies against PDL1, a frequently used cancer target, using our Beacon single Plasma Cell cloning platform as well as hybridoma technology. PD-L1 is a key inhibitor of T cell activation that is often over-expressed in cancer to escape immune surveillance and promote tumor progression. Blocking antibodies against PD-L1 or its receptor, PD-1, have shown significant clinical benefit in some patients with PD-L1 expressing tumors.

Hence, there is great interest in generating therapeutic antibodies against these targets to counteract the immune suppression mechanism that tumors rely on for survival. Most of the anti-PD-L1 therapies in the clinic have been generated by standard hybridoma technology. We investigated whether superior antibodies with greater diversity, affinity, and/or functional activity could be generated using single B cell cloning (BCC) which could circumvent a labor-intensive and time-consuming process. Thus, we generated unique antibodies against PD-L1 via both methods.

A detailed functional analysis of the obtained antibodies showed that the single cell cloning process using the Beacon platform generated less total hits than hybridoma, however, the antibodies showed better functionality.

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INTRODUCTION

Monoclonal antibodies (mAbs) against PDL-1 were rapidly developed by the ChemPartner Biologics group using Berkeley Lights’ Beacon® optofluidic single B-cell cloning technology. The high-throughput automation of the Beacon® system enabled us to identify PD-L1 blocking antibodies within a single day, significantly accelerating a process that would normally take 2–3 months using conventional methodologies such as hybridoma technology or synthetic display library.

Using the Beacon®, we screened tens of thousands of primary single plasma B cells to identify unique antibodies against PD-L1. A series of assays were used for direct screening on the Beacon®. These assays included bead-based binding/blocking assays, cell-based binding to CHOK1 cells overexpressing human PD-L1, and cell-based blocking assays using PD-1. Once positive hits were identified, single plasma B cells were exported by the Beacon for antibody sequence recovery. After single-cell reverse transcription, amplification, and sequencing by NGS, the sequence diversity of the anti-PD-L1 antibody hits was evaluated.

In addition, this technology can enable rapid comparison of target-specific repertoire diversity between different immunization strategies, mouse strains, and lymphoid organs. Through the rapid generation of diverse, high-affinity mAbs, clients can take advantage of this emerging technology to maximize their research success and advancement toward their commercial goals.

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INTRODUCTION

Several methodologies have been developed for generating monoclonal antibodies. Hybridoma technology is the most widely used method for antibody generation. However, there are major disadvantages to this process, such as being time consuming, laborious and of low-efficiency. Phage display, another common method used for antibody discovery, can often provide limited and biased repertoires due to non-cognate VH/VL pairing. Both of these methods have notable limitations, despite being important and reliable antibody discovery engines.

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