VHH, VHH-Fc, and bispecific nanobody

88 VHH nanobodies were generated from a naive llama VHH library and a humanized VHH nanobody library and selected for high-affinity binding to SARS-CoV-2 S protein through three rounds of S1 panning as described before (^{Dong et al., 2020}). ELISA was performed to examine the binding to S1 and RBD. Selected VHH nanobodies were further tested for their capability to block S protein binding to ACE2, DC-SIGN, L-SIGN, LSECtin, ASGR1, CLEC10A, and TTYH2, by cellular detection system (for ACE2, LSECtin, ASGR1, CLEC10A, and TTYH2) or ELISA (for DC-SIGN and L-SIGN). VHH-Fc was constructed by fusing VHH domain with human IgG1 Fc domain. Based on the results from competitive binding assay and blocking assay, two clones, A8 and G11, were picked to construct A8-G11-Fc bispecific nanobody. A8 and G11 were connected by a Gly-Ser linker. All the Fc-fusion VHH nanobodies were expressed in Expi293F cell and purified on a Protein A column.

To examine the blocking of S protein binding in the presence or absence of VHH nanobody, HEK293T cells were transfected with individual receptors as described above. 10 ng of S1-hFc (KACTUS Biosystems) (to check the binding to ACE2, ASGR1, CLEC10A and LSECtin) or RBD-hFc (KACTUS Biosystems) (to check the binding to TTYH2) were incubated with 50 μL VHH (various amount from 10 μg to 400 μg) overnight at 4°C to allow the binding of VHH to S protein and then added to each well in a 384-well plate. 15 ng of AF647-conjugated anti-human IgG secondary antibody (Invitrogen) were added to each well and incubated for 24 hr. Data was then collected by CDS and analyzed by CellProfiler software.

To examine the blocking of S protein binding in the presence or absence of human Fc-tagged VHH nanobodies, HEK293T cells were transfected with individual receptors as described above. 15 ng of S1-mFc (Sino Biological) or RBD-mFc (Sino Biological) (to check the binding to TTYH2) were incubated with 750 ng of VHH-hFc overnight at 4°C to allow the binding of VHH-hFc to S1-mFc or RBD-mFc and then added to each well in a 384-well plate. 20 ng of AF647-conjugated anti-mouse IgG secondary antibody (SouthernBiotech) was added to each well and incubated for 24 hr. Data was then collected by CDS and analyzed by CellProfiler software.