Broadly protective vaccines against known and pre-emergent human coronaviruses (HCoVs) are urgently needed. To gain a deeper understanding of cross-neutralizing antibody responses, we mined the memory B cell repertoire of a convalescent SARS donor and identified 200 SARS-CoV-2 binding antibodies that target multiple conserved sites on the spike (S) protein. A large proportion of the non-neutralizing antibodies display high levels of somatic hypermutation and cross-react with circulating HCoVs, suggesting recall of pre-existing memory B cells (MBCs) elicited by prior HCoV infections. Several antibodies potently cross-neutralize SARS-CoV, SARS-CoV-2, and the bat SARS-like virus WIV1 by blocking receptor attachment and inducing S1 shedding. These antibodies represent promising candidates for therapeutic intervention and reveal a target for the rational design of pan-sarbecovirus vaccines.

Broadly protective vaccines against known and pre-emergent coronaviruses are urgently needed. Critical to their development is a deeper understanding of cross-neutralizing antibody responses induced by natural human coronavirus (HCoV) infections. Here, we mined the memory B cell repertoire of a convalescent SARS donor and identified 200 SARS-CoV-2 binding antibodies that target multiple conserved sites on the spike (S) protein. A large proportion of the antibodies display high levels of somatic hypermutation and cross-react with circulating HCoVs, suggesting recall of pre-existing memory B cells (MBCs) elicited by prior HCoV infections. Several antibodies potently cross-neutralize SARS-CoV, SARS-CoV-2, and the bat SARS-like virus WIV1 by blocking receptor attachment and inducing S1 shedding. These antibodies represent promising candidates for therapeutic intervention and reveal a new target for the rational design of pansarbecovirus vaccines. In December 2019, a novel pathogen emerged in the city of Wuhan in China's Hubei province, causing an outbreak of atypical pneumonia (a disease known as COVID-19). The infectious agent was rapidly characterized as a lineage B betacoronavirus, named Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and shown to be closely related to SARS-CoV and several SARS-like bat CoVs(1). Despite the urgent need, there are currently no approved vaccines or therapeutics available for the prevention or treatment of COVID-19. Furthermore, the recurrent zoonotic spillover of CoVs into humans, along with the broad diversity of SARS-like CoV strains circulating in animal reservoirs, suggests that novel pathogenic CoVs are likely to emerge in the future and underscores the need for broadly active countermeasures.CoV entry into host cells is mediated by the viral S glycoprotein, which forms trimeric spikes on the viral surface(2). Each monomer in the trimeric S assembly is a heterodimer of S1 and S2 subunits. The S1 subunit is composed of four domains: an N-terminal domain (NTD), a C-terminal domain (CTD), and subdomains I and II(3-5). The CTD of both SARS-CoV and

Monovalent bispecific antibodies (BsAbs) are projected to have broad clinical applications due to their ability to bind two different targets simultaneously. Although they can be produced using recombinant technologies, the correct pairing of heavy and light chains is a significant manufacturing problem. Various approaches exploit mutations or linkers to favor the formation of the desired BsAb, but a format using a single common light chain has the advantage that no other modification to the antibody is required. This strategy reduces the number of formed molecules to three (the BsAb and the two parent mAbs), but the separation of the BsAb from the two monovalent parent molecules still poses a potentially difficult purification challenge. Current methods employ ion exchange chromatography and linear salt gradients, but are only successful if the difference in the observed isoelectric points (pIs) of two parent molecules is relatively large. Here, we describe the use of highly linear pH gradients for the facile purification of common light chain BsAbs. The method is effective at separating molecules with differences in pI as little as 0.10, and differing in their sequence by only a single charged amino acid. We also demonstrate that purification resins validated for manufacturing are compatible with this approach.