Development of multi-specific humanized llama antibodies blocking SARS-CoV-2/ACE2 interaction with high affinity and avidity

Abstract

Coronaviruses cause severe human viral diseases including SARS, MERS and COVID-19. Most recently SARS-CoV-2 virus (causing COVID-19) has led to a pandemic with no successful therapeutics. The SARS-CoV-2 infection relies on trimeric spike (S) proteins to facilitate virus entry into host cells by binding to ACE2 receptor on host cell membranes. Therefore, blocking this interaction with antibodies are promising agents against SARS-CoV-2. Here we describe using humanized llama antibody VHHs against SARS-CoV-2 that would overcome the limitations associated with polyclonal and monoclonal combination therapies. From two llama VHH libraries, unique humanized VHHs that bind to S protein and block the S/ACE2 interaction were identified. Furthermore, pairwise combination of VHHs showed synergistic blocking. Multi-specific antibodies with enhanced affinity and avidity, and improved S/ACE2 blocking are currently being developed using an in-silico approach that also fuses VHHs to Fc domains. Importantly, our current bi-specific antibody shows potent S/ACE2 blocking (KD - 0.25 nM, IC100 ∼ 36.7 nM, IC95 ∼ 12.2 nM, IC50 ∼ 1 nM) which is significantly better than individual monoclonal VHH-Fcs. Overall, this design would equip the VHH-Fcs multiple mechanisms of actions against SARS-CoV-2. Thus, we aim to contribute to the battle against COVID-19 by developing therapeutic antibodies as well as diagnostics.

Keywords: COVID-19; SARS-CoV-2; bi-specific antibody; humanized antibody; llama antibody; nanobody; tri-specific antibody.

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, K_on and K_off 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).