The importance of antibody research is self-evident, particularly as more companies delve into the development of antibody therapeutics following the COVID-19 pandemic. However, in the process of antibody drug development and industrialization, there exist several technical challenges and bottlenecks across multiple levels, such as:
- Difficulty in Target Identification: Identifying antibodies that can highly specifically bind to certain target proteins, especially those with complex structures or multiple epitopes, is often challenging.
- Balancing Affinity and Specificity: While high-affinity antibodies can more effectively bind to targets, excessively high affinity may increase the risk of adverse reactions.
- Immunogenicity Issues: Some antibodies may induce immune responses in the human body, leading to their recognition and clearance by the immune system, thereby reducing therapeutic efficacy. Addressing this issue requires designing safer antibody molecules.
- Production Process Complexity: Achieving scalable, efficient, and economical antibody production is a key technological challenge, including improving the expression levels of cell lines, optimizing cell culture processes, and reducing costs while increasing yield.
- Limitations of High-Throughput Screening Technologies: Despite existing mature screening technologies, selecting antibodies with desired properties from large antibody libraries remains a time-consuming and costly process.
- Cost and Time: The discovery and development of antibodies involve complex experimental designs and significant resource investment, typically taking several years or even more than a decade from candidate antibody discovery to clinical application, with high costs.
Today, we're excited to discuss a recently published article in the prestigious journal Nat Commun. titled "Accelerating Antibody Discovery with a Rapid Cell-Free Expression and Screening Platform." Our aim is to offer fresh perspectives and valuable insights for antibody developers.
Introduction
The study developed a rapid cell-free expression and screening platform for antibody discovery. By integrating cell-free DNA template generation, cell-free protein synthesis, and antibody fragment binding measurement technologies, the research team significantly shortened the evaluation time from several weeks to just a few hours. This process enables a single researcher to express and analyze the antigen-specific binding of hundreds of antibodies in less than 24 hours. Staff applied this workflow to analyze 135 previously published antibodies targeting the SARS-CoV-2 spike glycoprotein and successfully identified 8 highly effective neutralizing antibodies, which have been granted Emergency Use Authorization (EUA) by the US Food and Drug Administration (FDA) for the treatment of COVID-19. Additionally, 119 antibodies derived from immunized mice against the SARS-CoV-2 spike glycoprotein were screened, and several candidate neutralizing antibodies were identified.
Results
Development of a Workflow for Cell-Free DNA Assembly and Amplification
This process includes two key steps: first, using the Gibson assembly method to splice double-stranded linear DNA encoding the desired heavy chain variable region (VH) and light chain variable region (VL), followed by direct PCR amplification of the unpurified Gibson assembly product to generate linear expression templates (LET). This process can be completed within 3 hours in a 384-well plate without cell culture, demonstrating high efficiency and flexibility.
CFPS System for Antibody Fragment Expression
The team developed a crude extract-based cell-free protein synthesis system for rapid expression of antibody fragments. The system is based on the E. coli OrigamiTM B(DE3) strain carrying mutations in the trxB and gor genes, which allows for disulfide bond formation in the cytoplasm. By pretreating crude extract with a reducing agent inhibitor and supplementing specific E. coli enzymes, full-length trastuzumab (anti-HER2 antibody model) expression was successfully achieved from linear DNA templates. Given the optimization requirements for full antibody assembly, the study focused on synthesizing dimerized antigen-binding fragments (sdFab) instead, demonstrating their assembly consistency in the CFPS system compared to Fab fragments.
Evaluation of Antibody Fragment Binding and Assembly
After DNA assembly and CFPS, antigen-specific binding was evaluated. An AlphaLISA method was developed to assess direct binding from the CFPS reaction. AlphaLISA is a wash-free, high-throughput, and crude extract-compatible detection method that captures and quantifies interactions between target proteins and antibody fragments. After optimizing CFPS conditions, researchers used AlphaLISA to establish a strategy for measuring direct antigen binding, competitive binding, crosslinking, and protein complex formation. To validate the ability of AlphaLISA to profile neutralizing antibodies, four different commercial antibodies were tested for their ability to compete with ACE2 for binding to the SARS-CoV-2 receptor-binding domain (RBD). The determined IC50 values ranked similarly to the reported ELISA IC50s. Additionally, an sdFab assembly screen was developed using AlphaLISA to monitor antibody fragment expression and assembly in CFPS.
Evaluation of Previously Published Antibodies in Bulk
Using the in-house developed cell-free antibody expression and screening platform, the study evaluated 115 previously published antibodies targeting SARS-CoV-2. To ensure the robustness of the workflow, antibody fragments were expressed and evaluated in triplicate. Overall, these data indicate that the workflow can be used to express and evaluate human antibody fragments as a filter for selecting potential candidates for further development.
Expression and Evaluation of Highly Studied SARS-CoV-2 Antibodies
Subsequently, the study expressed and evaluated 8 antibodies previously granted Emergency Use Authorization (EUA) by the FDA for the prevention/treatment of COVID-19, as well as 11 other antibodies. Using AlphaLISA technology, the team detected the binding affinity of these antibody fragments to S6P, RBD, and competitive binding with ACE2, consistent with literature reports, accurately identifying the most effective neutralizing antibodies. Particularly for newly emerged concerning variants, the binding characteristics of these antibodies were also analyzed, demonstrating broad cross-reactivity against various SARS-CoV-2 variants and other coronaviruses. This workflow effectively validates its value in rapidly screening and identifying potential antibody candidates.
Discovery of SARS-CoV-2 Antibodies from Immunized Mice
The research team explored antibodies produced by immunized mice against the SARS-CoV-2 spike protein using the cell-free screening platform. Activated B cells positive for the Spike protein were isolated using fluorescence-activated cell sorting technology, and their sequences were sequenced, resulting in the screening of 119 different antibody fragments. Through AlphaLISA technology, the team comprehensively evaluated the binding ability of these antibody fragments to S6P, RBD, and ACE2, as well as sdFab assembly, showing good experimental reproducibility. Ultimately, this platform successfully identified a series of candidate antibodies with neutralizing potential, among which the antibody SC2-3 exhibited broad binding ability to all tested SARS-CoV-2 variants. These data suggest that the developed workflow can be used to discover antibodies targeting specific antigens.
Conclusion
The research introduces a groundbreaking cell-free platform for antibody expression and screening, drastically reducing the time required for antibody discovery and assessment from traditional weeks to mere hours. Through the integration of cell-free DNA template generation, protein synthesis, and AlphaLISA protein interaction detection, a comprehensive process has been developed for swiftly screening and evaluating antibody fragments targeting diverse antigens like SARS-CoV-2.
Designed for automation and high-throughput operations, the cell-free workflow features straightforward steps, facilitated by standardized liquid handling and temperature-controlled incubation. While manual intervention is currently necessary, its uncomplicated structure sets the stage for seamless end-to-end automation in the future. The cell-free protein expression platform exhibits immense potential for large-scale antibody screening, empowering researchers to rapidly identify and explore effective antibody therapies against future pandemics or other diseases.
In summary, this research offers an innovative solution to the challenges of antibody discovery and evaluation, streamlining the process for greater efficiency. By leveraging the capabilities of cell-free technology, researchers can expedite the development of therapeutic antibodies, providing optimism for tackling emerging infectious diseases and enhancing patient outcomes globally.
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