(W1030-02-06) Antibody Synthesis in Cell-Free for High-Throughput Production and Screening

Purpose: Antibody production is well established in cell-based platforms; however these are time consuming and costly processes¹. As an alternative, cell-free protein synthesis (CFPS) systems have been used to express immunoglobins and antibody fragments². Naturally antibodies are folded in periplasmic conditions in E.coli, which is eliminated when using CFPS³. Therefore, replication of periplasmic environment is required in order to successfully produce antibodies using CFPS systems. Different additives need to be optimized for each antibody, which makes the process time consuming. This project aims to develop a platform for expedited prototyping and screening of antibodies utilizing an E.coli lysate based CFPS system. This kind of platform can be further used to expedite the screening process of antibody libraries.

Methods: The proposed platform is based on our in-house made CFPS reactions, to which we add the linear DNA protection Tus-Ter system⁴ to prevent DNA degradation (which was developed in our lab⁵) and disulfide-bond supporting additives (glutathione⁶, DsbC⁷, PDI⁸, Erv1p⁹). The CFPS reactions are set-up in a multi-well manner followed directly by western blot and ELISA assay to identify properly folded antibodies with target binding affinity and high yield. 
For the conjugation experiments we use the SpyTag/SpyCatcher¹⁰ system and demonstrate immunofluorescence of HER2 positive cells.

Results: The conclusions from the preliminary experiments helped to define the necessary additives to support disulfide bond formation for proper antibody folding. Selected conditions were applied to express 38 antibodies (8 modalities, 6 targets) in CFPS system. It has been observed that the choice of lysate has a greater effect on successful antibody expression than other additives such glutathione or chaperones.
We recognized an initial set of 30 conditions as a good starting point for every antibody. The high-throughput platform was tested for three anti-HER2 antibodies: scFv (small), scFab (medium), scFv-Fc (large). The platform capabilities will be followed with anti-TNFα antibody library screen.
Furthermore, we have utilized our ability to express antibodies and demonstrated conjugation of fluorescent payload (YFP) to CFPS-antibodies, based on SpyTag/SpyCatcher system,  which is being confirmed in an immunostaining assay with HER2 positive cells.

Conclusion: Cell-free systems have been successfully used to produce antibodies and antibody fragments, but currently there is no universal system for expression of complex proteins like antibodies. Since the CFPS reactions require different combinations of additives to support disulfide bond formation, it is difficult and time consuming to identify the optimal conditions for each antibody of interest. To address this issue, a high-throughput platform for accelerated screening of CFPS reactions composition will allow to obtain the optimal expression condition for each antibody of interest. Once optimal condition is established, the reaction can be scaled-up and used as antibody production platform and be further expanded for payload conjugation. This kind of platform can be further used to expedite the screening of antibody libraries.

References: (1) Lee, Y. J. & Jeong, K. J. Challenges to production of antibodies in bacteria and yeast. Journal of Bioscience and Bioengineering vol. 120 483–490 Preprint at https://doi.org/10.1016/j.jbiosc.2015.03.009 (2015).
(2) Stech, M. et al. Cell-free synthesis of functional antibodies using a coupled in vitro transcription-translation system based on CHO cell lysates. Sci Rep 7, 12030 (2017).
(3) Berkmen, M. Production of disulfide-bonded proteins in Escherichia coli. Protein Expression and Purification vol. 82 240–251.
(4) Mulcair, M. D. et al. A Molecular Mousetrap Determines Polarity of Termination of DNA Replication in E. coli. Cell 125, 1309–1319 (2006).
(5) Norouzi, M., Panfilov, S. & Pardee, K. High-Efficiency Protection of Linear DNA in Cell-Free Extracts from Escherichia coli and Vibrio natriegens. ACS Synth Biol 10, 1615–1624 (2021).
(6) Chakravarthi, S., Jessop, C. E. & Bulleid, N. J. The role of glutathione in disulphide bond formation and endoplasmic-reticulum-generated oxidative stress. EMBO Rep 7, 271–275 (2006).
(7) Chen, J. et al. Chaperone activity of DsbC. Journal of Biological Chemistry 274, 19601–19605 (1999)
(8) Alanen, H. I. et al. Defining the Domain Boundaries of the Human Protein Disulfide Isomerases. Antioxid Redox Signal 5, 367–374 (2003).
(9) Hatahet, F., Nguyen, V. D., Salo, K. E. H. & Ruddock, L. W. Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of E. coli. Microb Cell Fact 9, 67 (2010).
(10) Zakeri, B. et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proceedings of the National Academy of Sciences 109, E690–E697 (2012).

Acknowledgements: Dr. Lia Cardarelli, Dr. Maryna Gorelik, Dr. Sadchev Sidhu.

Funding: Medicine by Design

Figure 1. High-throughput antibody screening in CFPS platform pipeline. The screening process of optimal disulfide bond conditions is expected to take only 2 days. Day 1: reaction setup in a 96 well PCR plate, overnight. Day 2: sample reactions are taken to qualitative western blot assay in parallel to quantitative ELISA.

Figure.2. Disulfide bond condition screen of anti-HER2 scFab antibody expressed in cell-free. Western blot and indirect ELISA of anti-HER2 scFab reactions to demonstrate expression of binding antibodies.

Figure 3. (Left) Western blot of anti-HER2 scFv-SpyTag cell-free reaction before and after conjugation to purified YFP-SpyCatcher. (Right) Immunofluorescence of HER2 positive cells with commercial Trastuzumab-Alexa 488 vs cell-free anti-HER2 scFab-YFP