Table 1 Intercomparison of multiple VHH-B11-derived conformations.

Name	E. coli	P. pastoris	HEK293F	Affinity	Linker1; linker2; linker3	Relative expression level	Configuration diagram	Gel photo
VHH	✓	✓	✓	~ 4.4 nM	NA	High	Figure 3B	Figure 4, lane1
VHH-VHH		✓	✓	~ 1.7 nM	(G4S)3	High	Figure 3C	Figure 4, lane2
VHH-VHH-VHH			✓	~ 0.01 nM	(G4S)3; (G4S)3	High	Figure 3D	Figure 4, lane6
VHH-Fc		✓	✓	~ 0.6 nM	IgG4-hinge linker	High	Figure 3F	Figure 4, lane7
VHH-CH1-Fc			✓	NA	NA; IgG4-hinge linker	Very low	Figure 3G	NA
VHH-VHH-Fc			✓	~ 0.8 nM	Llama long linker; IgG4-hinge linker	Medium	Figure 3H	Figure 4, lane8
VHH-VHH-CH1-Fc			✓	NA	(G4S)3; NA; hIgG4-hinge linker	Low	Figure 3I	Figure 4, lane9
VHH-Fc-VHH			✓	~ 0.6 nM	IgG4-hinge linker; Llama long linker	Medium	Figure 3J	Figure 4, lane10

Note: “VHH” represents single domain Ab VHH-B11. “✓” indicates that the Ab is expressed in this expression system. “-” represents the linker between the domains (VHH, CH1 or Fc). “(G4S)3” is a 15-peptide linker (amino acid sequence: GGGGSGGGGSGGGGS). “IgG4-hinge linker” indicates the hinge region between human Fab and human IgG4 Fc (amino acid sequence: PPCPSCPAPEFLGGPS); “Llama long linker” represents the hinge region between llama Fab and llama IgG2 Fc (amino acid sequence: EPKIPQPQPKPQPQPQPQPKPQPKPEPECTCPKCP). Relative expression level represents the expression status of each Ab in HEK293F cell expression system under same conditions. “High” means the purified Ab expression level > 20 mg/mL; “medium” means ~ 10 mg/mL; “low” means < 1 mg/mL; “very low” indicates that the Ab could be detected but cannot be purified easily; “NA” is not available.

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