This article is reprinted from the WeChat public account of "Shanghai ChemPartner".
With the wide application of antibody drugs and the huge social and economic benefits they generate, antibody drugs have gradually become an important member of the pharmaceutical field, and many domestic and foreign pharmaceutical companies have taken the research and development of antibody drugs as the company's key direction for strategic layout. Given the long cycle time and high failure rate required to move from basic research to real clinical translation, there will be more and more research and development for the same or similar targets, and the competition between pharmaceutical companies will become increasingly fierce. Therefore, it is crucial for pharmaceutical companies to efficiently develop antibody drug molecules with the best biological effects and druggability for the same target, which also puts forward higher requirements for antibody discovery platforms. Traditional hybridoma platforms require a lot of manpower and material resources due to multiple steps such as fusion, subcloning, and in vitro culture, and it usually takes several months to obtain ideal antibody candidates. Moreover, due to other factors such as the limitation of fusion efficiency, only a small percentage of antibody candidates will enter the subsequent screening stage, which limits the probability of obtaining antibody molecules with special properties. Antibody candidates obtained through the natural screening process in vivo usually have good physicochemical properties and low immunogenicity, which is conducive to later drug development and has its irreplaceable advantages. Therefore, the direct isolation of monoclonal antibodies from animals or humans remains a very important source.
In summary, how to efficiently and high-throughput screen the ideal antibody molecule from the natural antibody library puts forward higher requirements for antibody discovery technology platforms, and in order to cope with this development trend, some antibody R&D platforms based on innovative technologies have emerged.
At present, the antibody R&D platform based on the use of single B cell cloning mainly includes:
a) Single-cell sequencing technology platform based on flow cytometry sorting
Single B cells were sorted by flow cytometry, followed by in vitro culture screening and sequencing to obtain antibody sequences. However, the sorting process can cause certain mechanical damage to the cells and it is difficult to maintain the culture and screening of single cells in vitro, so it has high technical requirements and is limited by the throughput and efficiency of single-cell sorting, and the large-scale industrial application is not yet mature.
Fig.1 SARS-CoV-2 neutralizing antibody was obtained by flow cytometry
b) Microfluidics-based single-cell sequencing technology platform
Single B cells were sorted by microfluidic fluidics, and then the individual B cells were sequenced to obtain antibody sequences. The entire process is carried out in a high-throughput manner, enabling a large amount of sequence information to be obtained (Figure 2). However, it is usually not compatible with diversified functional experimental screening, and high-throughput plasmid extraction, cell transfection and screening are required in the later stage, resulting in large losses of labor and consumables, and limited efficiency.
Fig.2 SARS-CoV-2 neutralizing antibody was obtained using a microfluidic single-cell sequencing technology platform
The technology platform based on the Beacon single-cell lightguide system is an integrated antibody development system established by Berkeley Lights using its unique single-cell lightguide system, which deeply integrates semiconductor technology and the latest microfluidic technology, and can be compatible with various forms of functional screening, making it possible to conduct thousands of single-cell experiments at the same time. Berkeley Lights' OptoSelectTM chip overlays a perfusion-based nanofluidic system on an array of light-activated phototransistors, which makes it possible to quickly and precisely locate individual cells or other micro-objects using controlled light patterns. The OptoSelectTM chip contains thousands of flow-isolated nanocompartments known as NanoPensTM, which physically isolate cells from flow but allow for diffusion-based exchange. On the Beacon platform, thousands of single cells can be isolated, cultured, analyzed, and exported for further analysis using multiple OptoSelectTM arrays. With this system, precise manipulation of individual cells can be performed on a special chip, and various chip-based functional screenings can be performed, enabling the screening of antibody molecules with various biological activities in a relatively short time. Then, the cells that can secrete the ideal antibody molecule are exported through the light guide system and entered into the single-cell sequencing platform, so as to obtain the ideal antibody molecular sequence in one step. This system integrates complex multiple steps on a single platform, which can greatly shorten the development cycle and save a lot of material and manpower compared to other systems, greatly improving the efficiency of antibody drug discovery (Figure 3).
Figure 3 Introduction to Berkeley Lghts' Beacon technology platform
As a leading company in the biopharmaceutical CRO industry in China, ChemPartner signed a strategic cooperation agreement with Berkeley Lights in 2018 to develop the Beacon platform for antibody discovery. With the support of its existing integrated antibody discovery platform, after nearly three years of continuous development, ChemPartner has successfully added an innovative and integrated high-throughput antibody discovery platform in addition to the existing antibody discovery platforms such as hybridoma monoclonal and phage display. Today, ChemPartner has complementary antibody discovery platforms to meet the diverse antibody development needs of customers around the world (Figure 4).
Figure 4 Comparison of ChemPartner antibody discovery platforms
The following will introduce the Beacon antibody discovery platform in detail by taking PD-L1, a popular target in the field of tumor immunity, as a case study.
1. The Beacon antibody discovery platform can greatly shorten the antibody development cycle
Traditional hybridoma platforms require a lot of manpower and material resources due to multiple steps such as fusion, subcloning, and in vitro culture, and it usually takes several months to obtain ideal antibody candidates. The Beacon platform uses technologies such as OptoSelectTM chips to isolate, culture, analyze, and output thousands of single cells for further analysis, shortening the screening cycle to 1-2 days, thereby shortening the overall antibody discovery cycle by 3-4 months (Figure 5).
Fig.5 Comparison of the development cycles of Beacon single B cell clones and hybridomas
2. The Beacon antibody discovery platform combines multiplex experimental screening and high throughput
Although some traditional single-cell cloning-based technology platforms can also achieve high-throughput isolation and screening, the compatible experimental screening methods are often limited. Although a large number of monoclonal sequences can be obtained, it is often necessary to construct a large number of recombinant antibodies for further functional verification in the later stage, which greatly increases the cost and R&D cycle in the later stage. The Beacon antibody discovery platform can take into account both high-throughput and multiplex functional experimental screening, which can not only realize protein and cell-based binding experimental screening, but also take into account some functional antibody screening similar to ligand-receptor binding blocking and reporter gene function screening (Figure 6), so as to improve efficiency and shorten the development cycle.
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Fig.6 Multiple screening of Beacon single B cell cloning platform
3. The Beacon antibody discovery platform can explore a broader immune repertoire to increase antibody diversity and obtain high affinity and active molecule probability
Due to its high-throughput screening technology, the Beacon antibody discovery platform can screen a wider immune repertoire in a short period of time, and use multi-functional experimental screening to screen out highly active antibody molecules at an early stage, thus greatly improving the diversity of antibody drug molecules and the probability of obtaining highly active molecules. The resulting large number of antibody candidate molecules also poses great challenges for high-throughput characterization in the later stage. Carterra has developed the LSA high-throughput SPR detection system by integrating proprietary microfluidic technology and array SPR (surface plasmon resonance) detection technology, which can detect and analyze antibody affinity and binding epitopes at high throughput, greatly accelerating the antibody drug development cycle. Analysis of the results of PD-L1 cases combined with LSA high-throughput detection showed that the specific antibodies found through the Beacon platform exhibited comparable affinity and better blocking activity (Figure 7).
Figure 7 Comparison of antibody properties obtained by Beacon antibody discovery platform and hybridoma platform (high-throughput SPR technology)
In summary, compared with the hybridoma platform, the Beacon antibody discovery platform not only shortens the antibody discovery cycle, but also increases the success rate of obtaining antibodies with higher affinity and activity, thereby greatly improving the efficiency of antibody research and development (Fig. 8).
Fig.8 Comparison between the Beacon antibody discovery platform and the hybridoma platform
As one of the earliest antibody discovery teams in China, the Biologics R&D Department of ChemPartner has been adhering to the concept of science first and technology-based, and has established a diversified and comprehensive antibody discovery platform, which can provide personalized and efficient antibody discovery services to customers around the world.