Bispecific antibodies combine breadth, potency, and avidity of parental antibodies to neutralize sarbecoviruses

Abstract

SARS-CoV-2 mutational variants evade humoral immune responses elicited by vaccines and current monoclonal antibody (mAb) therapies. Novel antibody-based treatments will thus need to exhibit broad neutralization against different variants. Bispecific antibodies (bsAbs) combine the specificities of two distinct antibodies into one antibody taking advantage of the avidity, synergy and cooperativity provided by targeting two different epitopes. Here we used controlled Fab-arm exchange (cFAE), a versatile and straightforward method, to produce bsAbs that neutralize SARS-CoV and SARS-CoV-2 variants, including Omicron and its subvariants, by combining potent SARS-CoV-2-specific neutralizing antibodies with broader but less potent antibodies that also neutralize SARS-CoV. We demonstrate that the parental IgG's rely on avidity for their neutralizing activity by comparing their potency to bsAbs containing one irrelevant "dead" Fab arm. We used single particle mass photometry to measure formation of antibody:spike complexes, and determined that bsAbs increase binding stoichiometry compared to corresponding cocktails, without a loss of binding affinity. The heterogeneous binding pattern of bsAbs to spike (S), observed by negative-stain electron microscopy and mass photometry provided evidence for both intra- and inter-spike crosslinking. This study highlights the utility of cross-neutralizing antibodies for designing bivalent or multivalent agents to provide a robust activity against circulating variants, as well as future SARS-like coronaviruses.

Broadening sarbecovirus neutralization with bispecific antibodies combining distinct conserved targets on the receptor binding domain

“…In addition, we have previously shown that bsAbs generated by cFAE retained in vitro antibody-dependent cellular phagocytosis and trogocytosis 18 , and expect the bsAbs described in this study to retain these properties as well. However, particularly mAbs that do not use direct ACE2 blocking as their mechanism of neutralization, often rely on S destabilization, conformational changes and Fc-mediated effector functions to combat viral infection 42 .…”

“…Different SARS-CoV-2 targeting bsAbs have been described, which show improved neutralization activity compared to the parental mAbs 18,20,21 . The so far described IgG-like bsAbs are of highly diverse formats, such as the CrossMAb constructs [22][23][24][25][26] , DVD-Ig 24,27 , IgG-(scFv)2 21,24,28,29 , Tandem scFv-Fc 28,30,31 , and VH/Fab IgGs 32,33 , many of which require significant engineering and quality control processes to be efficiently produced.…”

Overcoming antibody-resistant SARS-CoV-2 variants with bispecific antibodies constructed using non-neutralizing antibodies

“…Another approach is to investigate different antibody formats, bispecific or multispecific, that can simultaneously and synergistically bind to multiple epitopes. [42][43][44][66][67][68][69][70][71][72][73] A study on bispecific antibodies combining anti-RBD and anti-S2 antibodies has been reported. 44 The structure of these bispecific antibodies involved a combination of scFvs from neutralizing antibodies against RBD and S2, arranged in tandem in the scFv-scFv-Fc format.…”

Bispecific antibodies with broad neutralization potency against SARS-CoV-2 variants of concern

“…Previous studies have highlighted the effectiveness of bispecific constructs (41,(53)(54)(55)(56)(57)(58)(59)(60)(61)(62)(63)(64)(65)(66)(67)(68), including one notable example where constructs paired broadly cross-reactive weak or non-neutralizing mAbs with a potent inhibitor of viral entry, which significantly enhanced its overall neutralization potency and breadth (55). Given C1596's broad cross-reactivity against SARS-CoV-2 VOCs and trapping of RBDs in an "up" state, we investigated the ability of C1596 to support bivalent interactions with an RBD-specific antibody in a single-molecule bispecific format (Supplemental Figure 6).…”

“…To address the diminished effectiveness of nAb monotherapies and therapeutic cocktails, several research groups, including ours, have developed multivalent constructs such as bispecific antibodies (bsAbs). These bsAbs feature two different antigen-binding fragments (Fabs) targeting non-overlapping spike epitopes (41,(53)(54)(55)(56)(57)(58)(59)(60)(61)(62)(63)(64)(65)(66)(67)(68) and exhibit increased resilience against viral evasion compared to monoclonal antibodies, akin to antibody cocktails (69,70). In addition, bsAbs offer a distinct advantage by being manufactured for delivery as a single molecule and have the potential to leverage potential avidity-driven intra-spike and inter-spike crosslinking (54,68).…”