A “Super助攻” in Nanobody Development: Cell-Free Protein Synthesis (CFPS) Technology-PLD_biomedical

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A “Super助攻” in Nanobody Development: Cell-Free Protein Synthesis (CFPS) Technology

Release date:

2024-06-06

　　Today, biomedical research is advancing into the subcellular realm, driven by the rapid progress of nanomedicine, nanodiagnostics, and nanotherapy. Meanwhile, nanoparticles are being widely employed in disease prevention, diagnosis, treatment, and follow-up monitoring. Nanobodies, as a novel and distinctive class of antigen-binding fragments, are emerging as a popular choice for developing next-generation biologics, owing to their advantageous properties: small size, high stability, strong antigen‑binding affinity, excellent water solubility, and natural origin. At present, several nanobodies have entered clinical trials, opening up new avenues for the treatment of diverse human diseases. However, the screening and functional validation of nanobodies remain laborious and time‑consuming. In recent years, the rapid advancement of cell‑free protein synthesis (CFPS) technology has ushered in fresh opportunities for the discovery and development of nanobodies.

　　I. Nanobodies: Screening and Functional Validation

　　Nanobodies (Nbs), also known as single-domain antibody fragments derived from the heavy-chain variable region of antibodies, are antibody fragments originating from the heavy-chain‑only IgG antibodies found in camelids. Over the years, nanobodies have attracted considerable attention across diverse research fields, particularly in disease diagnosis and therapy. The world’s first nanobody‑based drug, caplacizumab, was approved in 2018, followed shortly thereafter by additional approvals. Compared with conventional antibodies, nanobodies offer several advantages: 1) enhanced specificity and tissue penetration; 2) superior stability, including resistance to high temperatures; 3) greater suitability for large‑scale industrial production; and 4) easier engineering and optimization.

Figure 1: Schematic diagram of the methods for generating nanobodies, their characterization, and potential applications.

　　The screening and functional validation of nanobodies are critical steps in the nanobody development process. Screening enables the selection of antibodies with high affinity and specificity from a large pool of candidates, while functional validation is essential to ensure that the chosen antibodies can deliver the intended performance in practical applications. Both processes require extensive protein expression and purification; therefore, a rapid and efficient protein expression system is indispensable for accelerating nanobody research and development.

Figure 2: Flowchart of nanobody-based diagnostics

　　II. Advantages of CFPS Technology in Nanobody Screening and Functional Validation

　　Compared with conventional intracellular expression systems, the CFPS system offers numerous advantages in the screening and functional validation of nanobodies:

　　1. It can produce large quantities of target proteins in an extremely short time, significantly shortening the experimental timeline. While conventional protein expression typically takes 2 to 3 days, the CFPS system can reduce this timeframe to just 1 to 2 hours.

　　2. It can achieve high‑yield protein synthesis without involving complex intracellular regulatory mechanisms, making it easier to optimize reaction conditions and thereby enhancing both the expression level and quality of specific proteins.

　　3. It supports high-throughput screening, enabling researchers to rapidly identify nanobodies with potential therapeutic value from vast libraries of candidate antibodies within a short timeframe. This approach is particularly well-suited for the initial screening of nanobodies, significantly reducing both experimental time and costs.

　　4. In terms of functional validation, because cell-free systems are open reaction platforms, researchers can more easily control experimental conditions and conduct detailed assays of biological activities, such as affinity, stability, and specificity. This is crucial for ensuring the efficacy and safety of nanobodies in practical applications.

Figure 3: General process of cell-free production

　　In summary, cell-free protein expression technology offers numerous advantages for the screening and functional validation of nanobodies, including high efficiency, high yield, high throughput, and easily controllable experimental conditions. These benefits have significantly accelerated the development process and improved the success rate of nanobody research, making it an indispensable tool in modern biopharmaceutical studies.

　　III. Case Sharing

　　In recent years, an increasing number of studies have demonstrated the substantial potential of CFPS in nanobody development. Krebs SK, Stech M, and colleagues reported that active recombinant immunotoxins (RITs) can be produced in both Escherichia coli and CHO cell-free systems, and they showed that CFPS enables on-demand assessment of antibody–toxin binding activity within a time‑efficient workflow, without the need for cell lysis or purification.

Figure 4: Quantitative analysis, self‑crosslinking, and antigen binding of proteins produced in a cell‑free system using Escherichia coli, in the presence of the GroEL/GroES chaperonin.

　　In another study, Ding R, Hung KC, and colleagues employed a hybridoma cell library to characterize the system, utilized droplet-based sorting, and integrated CFPS technology to generate single-chain variable fragment (scFv) antibodies from 14 sorted cell lines. Among these, 12 exhibited antigen-specific binding as assessed by ELISA.

Figure 5: Analysis of Isolated Antibody Sequences

　　IV. Conclusion

　　The rapid advancement of CFPS technology has brought significant convenience to the screening and functional validation of nanobodies. Its rapid expression capability not only shortens the R&D cycle but also enhances the efficiency and accuracy of screening. As this technology continues to be optimized and refined, it is expected that cell-free expression systems will play an even more pivotal role in the biopharmaceutical field—particularly in the development of nanobodies—thereby contributing further to human health.

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　　References:

　　Jovčevska I, Muyldermans S. The Therapeutic Potential of Nanobodies. BioDrugs. 2020;34(1):11-26. doi:10.1007/s40259-019-00392-z.

　　Jin BK, Odongo S, Radwanska M, Magez S. Nanobodies: A Review of Generation, Diagnostics and Therapeutics. Int J Mol Sci. 2023;24(6):5994. Published 2023 Mar 22. doi:10.3390/ijms24065994.

　　Muyldermans S. Applications of Nanobodies. Annu Rev Anim Biosci. 2021;9:401-421. doi:10.1146/annurev-animal-021419-083831.

　　Krebs SK, Stech M, Jorde F, et al. Synthesis of an Anti-CD7 Recombinant Immunotoxin Based on PE24 in CHO and E. coli Cell-Free Systems. Int J Mol Sci. 2022;23(22):13697. Published 2022 Nov 8. doi:10.3390/ijms232213697.

　　Dondapati SK, Stech M, Zemella A, Kubick S. Cell-Free Protein Synthesis: A Promising Option for Future Drug Development. BioDrugs. 2020;34(3):327-348. doi:10.1007/s40259-020-00417-y.

　　Ding R, Hung KC, Mitra A, et al. Rapid isolation of antigen-specific B-cells using droplet microfluidics. RSC Adv. 2020;10(45):27006-27013. Published 2020 Jul 20. doi:10.1039/d0ra04328a.