Four nanobodies (A31, B13, B18, and C2) from groups A and B showed effective neutralizing activity against subvariants BA.1 and BA.4/5, but completely lost their ability to neutralize subvariants BQ.1.1 and XBB.1, suggesting that they target nonconserved epitopes on the RBD. and XBB.1. To Butylscopolamine BR (Scopolamine butylbromide) enhance the therapeutic potential, we engineered a panel of multivalent nanobodies with increased neutralizing potency and breadth. The most potent multivalent nanobody, B13-B13-B13, cross-neutralized all tested pseudoviruses, with a geometric mean of the 50% inhibitory concentration (GM IC50) value of 20.83 ng/mL. An analysis of the mechanism underlying the enhancement of neutralization breadth by representative multivalent nanobodies demonstrated that the strategic engineering approach of combining two or three nanobodies into a multivalent molecule could improve the affinity between a single nanobody and spike, and could enhance tolerance toward escape mutations such as R346T and N460K. Our engineered multivalent nanobodies may be promising drug candidates for treating and preventing infection with Omicron subvariants and even future variants. Keywords: SARS-CoV-2, Omicron subvariants, phage display library, receptor-binding domain (RBD), multivalent nanobodies, neutralizing activity 1. Introduction A beta coronavirus known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is to blame for the unprecedented coronavirus disease 2019 (COVID-19) pandemic [1,2]. As of 21 October 2023, the global tally of confirmed COVID-19 cases surpassed 771 million, with a staggering death toll of 6.9 million individuals (https://covid19.who.int/ (accessed on 21 October 2023)). Moreover, this unprecedented pandemic has profoundly disrupted economies and strained healthcare systems worldwide. The scientific community has been diligently striving to develop effective treatments and vaccines against SARS-CoV-2. Therapeutic neutralizing antibodies represent a critical class of antiviral treatments that have been rapidly developed and deployed within a short period for the prophylaxis and treatment of SARS-CoV-2 [3]. However, the ongoing adaptive evolution of SARS-CoV-2 has resulted in the emergence of several variants of concern (VOCs), which pose a significant challenge to the effectiveness of antibody treatments. This is particularly true for Omicron and its descendant subvariants, as they have not only impaired the efficacy of most neutralizing antibody treatments, but also undermined the effectiveness of existing vaccines [4,5,6,7]. The original Omicron variant (B.1.1.529) was first detected in South Africa toward the end of 2021 and quickly gained worldwide attention as it rapidly spread across different countries and continents [8]. Compared to other VOCs, Omicron exhibits higher transmissibility and stronger immune evasion due to the presence of numerous mutations within the spike (S) protein [4,9,10]. Over time, Omicron has expanded into multiple subvariants, such as BA.1, BA.2, BA.2.75, BA.4 and BA.5 (hereafter BA.4/5, as these two share an identical spike sequence), BF.7, BQ.1.1 and XBB; these subvariants have further exacerbated public health concerns. In particular, BA.4/5 subvariants can further evade acquired immunity developed from previous infections with BA.1 or BA.2 subvariants, resulting in a surge of breakthrough infections in numerous regions globally [11,12]. Subsequently, novel subvariants BQ.1.1 and XBB.1 have emerged and supplanted BA.5 as the prevailing variants due to their stronger ability to evade antibodies [13,14]. Notably, all clinically authorized therapeutic antibodies were rendered inactive against subvariants BQ.1.1 and Butylscopolamine BR (Scopolamine butylbromide) XBB.1 [13]. Moreover, a BA.5 bivalent booster was found to fail to elicit a robust immune response against BQ.1.1 and XBB.1, further highlighting the remarkable antibody evasion properties of these two subvariants [15]. Therefore, it is imperative to develop potent and broad-spectrum neutralizing antibodies for the effective management of Omicron subvariants and proactive preparedness against future emerging variants. The spike (S) glycoprotein of SARS-CoV-2, which is composed of the S1 and S2 subunits, plays a crucial role in infecting host cells [16,17,18]. The S1 subunit can be divided into an N-terminal domain (NTD) and a receptor-binding domain (RBD) [18]. Within the RBD, there is a receptor-binding motif (RBM) responsible for recognizing and binding to a specific host cell receptor known Butylscopolamine BR (Scopolamine butylbromide) as angiotensin-converting enzyme 2 (ACE2) [19,20]. Once bound to ACE2, the S2 subunit facilitates viral fusion with host cell membranes [21,22]. Rabbit Polyclonal to NUMA1 Based on this infection process, the spike glycoprotein is considered an effective target for neutralizing antibody therapy. The current classification of neutralizing antibodies against the spike glycoprotein includes three distinct Butylscopolamine BR (Scopolamine butylbromide) categories: those that target the NTD, those that target the RBD, and those that target the S2 subunit [23]. The majority of the highly potent neutralizing antibodies developed thus far have predominantly focused on targeting the RBD [23,24,25]. Their RBD epitopes are grouped into seven core communities, which are located on the cryptic inner face, the solvent-exposed outer face, and the top RBM face of the RBD [26]. Neutralizing antibodies that specifically bind to the RBM can directly inhibit the interaction between ACE2 and.