Title: 1 Simultaneous evaluation of antibodies that inhibit SARS-CoV-2 RBD variants 2 with a novel competitive multiplex assay. 3

37 The SARS-CoV-2 Receptor Binding Domain (RBD) is both the principal target of 38 neutralizing antibodies, and one of the most rapidly evolving domains, which can result 39 in the emergence of immune escape mutations limiting the effectiveness of vaccines 40 and antibody therapeutics. To facilitate surveillance, we developed a rapid, high- 41 throughput, multiplex assay able to assess the inhibitory response of antibodies to 24 42 RBD natural variants simultaneously. We demonstrate that immune escape can occur 43 through two mechanisms, antibodies that fail to recognize mutations, along with 44 antibodies that have reduced inhibitory capacity due to enhanced variant RBD-ACE2 45 affinity. A competitive approach where antibodies simultaneously compete with ACE2 46 for binding to the RBD may therefore more accurately reflect the physiological 47 dynamics of infection. We describe the enhanced affinity of RBD variants N439K, 48 S477N, Q493L, S494P and N501Y to the ACE2 receptor, and demonstrate the ability 49 the ACE2-RBD interaction. Here we tested two commercially available neutralizing antibodies (one human and one mouse), and two negative controls for RBD binding and ACE2 inhibition. The first negative control was an unrelated human