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. 2020 May 22;9(1):1034–1036. doi: 10.1080/22221751.2020.1768806

Figure 1.

Figure 1.

The workflow of anti-SARS-CoV-2 antibody discovery. (1) PBMC from 65 llamas were obtained, RNA was isolated, and cDNA was generated. Then, the VHH genes were amplified by two rounds of PCR and cloned to a phage display vector to construct the naïve VHH library. The synthetic VHH library was prepared by incorporation of shuffled VHH CDR1, 2 and 3, generated by overlapping PCR, to a modified human VH scaffold. (2) The VHH phage libraries were used for panning SARS-CoV-2 S1 fused to mouse Fc protein as the target antigen. Wells were coated with anti-mouse Fc to immobilize the antigen, and 3 rounds of phage panning were performed with reduced antigen concentration in each round. In ELISA assays, plates were coated with SARS-CoV-2 S1, the bound VHHs were detected by biotinylated anti-c-Myc antibodies and subsequent addition of streptavidin-HRP. The phylogenetic tree for 69 unique VHH binders is shown. (3) ELISA for ACE2 competition assay was performed by coating the plates with SARS-CoV-2 S1 as described previously and adding VHH in the presence of biotinylated ACE2. S1/ACE2 blocking function was determined by the reduction of HRP-induced chemiluminescence signal. The list of 9 unique S/ACE2 blockers is shown. (4) The ACE2 competition assay was repeated with a pairwise combination of the 9 S/ACE2 blockers, and the results are shown. (−): >=100%, (+): 80%−100%, and (++): <80% of the signal remaining compared to single VHH additions. Two VHH pairs have synergistic effects on blocking as shown in Red. (5) Structural organization of bi-specific and tri-specific llama VHH nanobody-Fc molecules that have been designed. The design process utilizes CAAD that optimizes features of VHH-Fcs. The concentration-dependent blocking of S/ACE2 binding by monoclonal (1B and 3F) and bi-specific (1B-3F) VHH-Fcs, and their IC100 differences are shown. The KD, Kon and Koff values for S protein binding by those antibodies are also shown. (6) Potential therapeutic mechanisms of ABS nanobody-Fcs. (7) Potential diagnostic utilization of humanized llama VHHs as single or combinatorial probes. (Created with BioRender.com).