The coding fragment of ACE2, which fused having a GP67 signal peptide at N terminus to facilitate protein secretion and a 6His tag at C terminus for protein purification, was also inserted into pFastBac1 vector for protein preparation. and Nb-021) that target non-overlapping epitopes in SARS-CoV-2 S-RBD with high affinity. Both nanobodies could broadly neutralize varied SARS-CoV-2 variants. Furthermore, we exposed two unique neutralization mechanisms of Nb-015 and Nb-021 by high-resolution nanobody/S-RBD complex constructions. Eventually, we designed MLN 0905 a bispecific nanobody conjugate which exhibits superb inhibitory activity against SARS-CoV-2, including the initial strain and the VOI and VOC variants. We believe that the nanobody conjugate recognized in this study could serve as an effective anti-viral drug reserve for potential treatment of COVID-19. == Intro == Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of coronavirus disease 2019 (COVID-19), offers led to a global pandemic enduring for over three years. To date, over 757 million people have suffered from the viral illness, leading to more than 6.8 million deaths worldwide (https://covid19.who.int/). SARS-CoV-2 in the beginning binds to the sponsor receptor, angiotensin-converting enzyme 2 (ACE2), from the receptor-binding website (RBD) of its spike (S) glycoprotein to tether the viral particles to the cell surface [1,2]. This receptor binding process is definitely believed to consequently result in the S-mediated membrane fusion, eventually leading to viral access to set up illness [3,4]. Consequently, the RBD of S (S-RBD) is definitely a major target of the sponsor humoral immunity [5]. Multiple antibodies that target SARS-CoV-2 S-RBD have thus far been developed to fight against the viral illness, and several of them have been authorized for medical treatment of COVID-19 [68]. However, the emergence of SARS-CoV-2 variants, especially those defined from the World Health Business as variants of interest (VOIs) or concern (VOCs), offers raised new difficulties because of their improved transmissibility and immune-escape capacity. Several RAC3 mutations recognized in the VOI and VOC strains are shown to seriously compromise the neutralizing effectiveness of available restorative antibodies [9,10]. Of unique note is the Omicron variant that has accumulated more than 30 mutations in S, among which over 15 are located in S-RBD. As a result, only a limited number of antibodies focusing on the CR3022 site or S309 site retain similar binding towards Omicron S-RBD, whereas antibodies focusing on the RBS-A, -B, -C and -D sites are significantly escaped from the computer virus [11,12]. Thus, there is an urgent and continuing need to determine antibodies able to counter all current SARS-CoV-2 variants including Omicron. Camelid-derived VHH antibodies, MLN 0905 also known as nanobodies, are natural, single-heavy-chain monovalent antibody fragments [13]. In comparison to standard antibodies, nanobodies symbolize the smallest antigen-binding website, consequently featuring with several physicochemical advantages, such as smaller size (12~15 kDa), less difficult production in different expression systems, higher thermal stability and solubility, etc [14]. In addition, nanobodies can be very easily bioengineered into homo/heterodimers or multimers, such as by fusing to an immunoglobulin-G (IgG) Fc tag or simply by linking two or more nanobodies inside a tandem manner, to increase the neutralizing potency and reduce the risk for viral escape [15,16]. These unique properties of nanobodies have led them to become developed mainly because potential therapeutics against viral illness, including SARS-CoV-2 MLN 0905 illness [17,18]. Here, we statement the recognition and characterization of two neutralizing nanobodies isolated from an immunized alpaca, Nb-015 and Nb-021, which target two non-overlapping epitopes on SARS-CoV-2 S-RBD. Structural and practical studies reveal MLN 0905 two unique neutralizing mechanisms for Nb-015 and Nb-021. The former recognizes a conserved epitope in the ridge of the S-RBD.