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Abstract
Antibodies are indispensable tools for basic research as well as diagnostic and therapeutic applications. Consequently, the development of alternative manufacturing strategies which circumvent the hurdles connected to conventional antibody production technologies is of enormous interest. To address this issue, we demonstrate the synthesis of complex antibody formats, in particular immunoglobulin G (IgG) and single-chain variable fragment Fc fusion (scFv-Fc), in a microsome-containing cell-free system based on translationally active chinese hamster ovary (CHO) cell lysates. To mimic the environment for antibody folding and assembly present in living cells, antibody genes were fused to an endoplasmic reticulum (ER)-specific signal sequence. Signal-peptide induced translocation of antibody polypeptide chains into the lumen of ER microsomes was found to be the prerequisite for antibody chain assembly and functionality. In this context, we show the rapid synthesis of antibody molecules in different reaction formats, including batch and continuous-exchange cell-free (CECF) reactions, depending on the amount of protein needed for further analysis. In addition, we demonstrate site-specific and residue-specific labeling of antibodies with fluorescent non-canonical amino acids. In summary, our study describes a novel antibody production platform which combines the highly efficient mammalian protein folding machinery of CHO cells with the benefits of cell-free protein synthesis.
Highlights
Due to their remarkable abilities as binding and detection reagent, antibodies have become indispensable tools for biomedical applications including the treatment of cancer, autoimmune and inflammatory disorders[1,2,3]
Co-expression of light chain (LC) and heavy chain (HC) in a 1:1 molar ratio resulted in the detection of LC monomer (~26 kDa), HC monomer (~55 kDa) and the assembled LC/HC heterotetramer (IgG) showing an apparent molecular weight (MW) of approximately 160 kDa. scFv-Fc was detectable as monomer (~60 kDa), and as homodimer with an apparent MW of 110 kDa
Protein synthesis based on linear DNA templates resulted in the assembly of immunoglobulin G (IgG) molecules as detected by autoradiography, identifying the PCR product ratio of 1:1 as the optimal one leading to the highest percentage of intact IgG (58%) (Supplementary Fig. 2b)
Summary
Due to their remarkable abilities as binding and detection reagent, antibodies have become indispensable tools for biomedical applications including the treatment of cancer, autoimmune and inflammatory disorders[1,2,3]. We anticipate that a technology that is able to accelerate the antibody screening phase during lead identification and optimization will be highly in demand To address this issue, we have developed a microsome-containing cell-free expression system based on CHO cells. Microsome containing eukaryotic cell-free systems lagged behind prokaryotic ones when it came to production yields but have caught up[24] In this context, a high-yield cell-free system based on CHO cell lysates has been developed in our lab, demonstrating the synthesis of functionally active membrane proteins and antibody fragments. We anticipate that this system will constitute a novel tool for the acceleration of antibody development and screening during lead identification and optimization
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