## 4 Discussion  
Full-sized antibodies have been used as therapeutic agents against immunological disorders. However, their large size leads to low tissue accessibility and penetration, thereby affecting their therapeutic efficacy [11]. Therefore, smaller engineered antibody fragments have become promising alternatives to full-sized antibodies [12]. ScFvs (approximately 30-35 kDa) and sdAbs (approximately 15-20 kDa) have inherent advantages over full-sized antibodies (approximately 150 kDa), including smaller sizes, relatively low production costs, and improved robustness, and have thus attracted considerable scientific attention and applications [13]. Therefore, potential antibody fragment-based cancer therapies using scFv and sdAbs against PD-LI developed in this study would be particularly important in biomedical and pharmaceutical fields. Moreover, for antibody engineering, sdAbs could enable the production of novel multivalent constructs, such as combination antibodies and bispecific antibodies. Furthermore, sdAbs are characterized by their unique biophysical features, e.g., pharmacological features, such as lower immunogenicity than scFv and alpaca-based VHH having a size similar to VH and VL. Despite the advantages of scFvs and sdAbs, some issues need to be addressed. First, they have a shorter half-life than Fab or full-sized antibodies. This could be improved by conjugating engineered Fc-based constructs, such as monomeric IgG CH3 or CH2-CH3 hybrids [14; 15; 16; 17; 18]. This strategy can rescue fused scFvs or sdAbs from endosomal degradation, although such fusion will result in an increase in the size. Second, the GS linker of scFv, which links VH and VL, could potentially incur some immunogenicity because the GS peptide is a non-native antibody sequence. Hence, it would be preferable to use VH or VL instead of scFv, despite the lower sensitivity of sdAbs than scFv. Lastly, future research should focus on exploiting sdAb affinities. Sensitivity-related biophysical properties can be exploited by introducing mutations in complementarity-determining regions followed by screening from libraries that allow a more hydrophilic VH-VL interface and/or higher affinity [19; 20]. Herein, we used _E. coli_ as host cells owing to the shorter growth time and to the less production cost than those of mammalian cells and yeasts. This allows rapid and  
Figure 3: (**A**) Schematic representation of the indirect ELISA for confirming the dose-dependent PD-L1-binding efficiency of recombinant antibody fragments; (**B**) ELISA signal of 100 ng anti-PD-L1 scFv with various concentrations PD-L1; (**C**) ELISA signal of 1000 ng anti-PDL1 VH with various concentrations PD-L1; (**D**) ELISA signal of 1000 ng anti-PD-L1 VL with various concentrations of PD-L1. Error bars represent \(\pm\)1 SD (n = 3). cost-effective production of antibody fragments in large quantities. In addition, the transformation of _E. coli_ with nanogram amounts of DNA by convenient heat shock treatment shows high efficiency in comparison to the transformation of mammalian cells, which requires expensive transfection reagent. _E. coli_ lacks subcellular compartments, such as olgi apparatus and endoplasmic reticulum, which are necessary to facilitate glycosylation [21]. However, as scFv and sdAb do not require glycosylation, whereas full-sized antibody is glycosylated at its hinge and Fc region, it was not an obstacle to produce the antibody fragments in _E. coli_, while full-sized antibody cannot be properly be formed via the _E. coli_-based expression system [22; 23; 24]. However, _E. coli_-based expression can trigger an endotoxic immune response that leads to toxic shock in humans and/or can induce contamination of the sample due to the presence of lipopolysaccharideaccharides (LPS) [21].