We proceeded to characterize the epitopes of Fab C01 and Fab D01 by sequential bio-layer interferometry , where one binder is incubated with the antigen until saturation, followed by incubation with the second binder. Robust binding of the second binder in the presence of the first suggests that the two binders occupy non-overlapping epitopes on the same antigen. Both Fab C01 and Fab D01 do not overlap with ACE2-Fc, indicating that they bind outside of the ACE2 binding site . Interestingly, while Fab C01 and D01 compete for binding on Spike-RBD, on Secto their epitopes appear to not overlap . Additionally, we observe that presaturation with Fab D01 blocks binding of Fab C01 on Secto, while pre-saturation with Fab C01 does not block Fab D01 . These data suggest that although C01 and D01 have overlapping epitopes on isolated Spike-RBD, these Fabs have different binding mechanisms and could be influenced by the dynamics and accessibility of the RBDs in the context of the Spike trimer. Therefore, we assign site C and site D to unique epitopes on Secto though they overlap on Spike-RBD. Additionally, we compared the epitopes of Fab C01 and Fab D01 to a previously described antibody CR3022,and found that Fab D01 competes with CR3022 while Fab C01 does not . CR3022 has been shown to recognize the RBD outside of the ACE2 binding site at an epitope only accessible when the RBD is in the “up” conformation.Fab D01 appears to bind an overlapping epitope,vertical farming supplies but with higher affinity to SpikeRBD-Fc compared to CR3022 . Thus, from these studies, we have identified binders against 4 unique epitopes on Spike-RBD: site A , site B , site C , and site D .
We wondered whether combining VH and Fab binders directed at different Spike epitopes into a bi-specific antibody scaffold could improve potency. One of the most straightforward and successful methods to make bi-specific IgG-type binders is the classic Knob-In-Hole strategy, pioneered by Paul Carter’s Group at Genentech.In this approach, a complementary setof mutations in the CH3 domains of the Fc promotes the heterodimerization between a “knob” Fc and a “hole” Fc. A KIH bi-specific IgG modality has not been reported for COVID-19 to date but has been previously developed for other viral infections such as HIV and showed improved potency compared to the mono-specific parental binders.Therefore, generating KIH bi-specifics against Spike could present a novel, effective modality against SARS-CoV-2. When the KIH strategy is applied for two Fab arms, the individual half IgGs must be separately expressed and purified before they can be assembled into a bi-specific IgG molecule due to the presence of two different light chains. This process is required to circumvent the problem of light chains and heavy chain mispairing that can occur when the antibodies are co-expressed in the same cell. While many strategies have been developed to try and address this issue, including using common light chains, additional purifications, or scaffold engineering, they all require additional labor-intensive steps.However, since the VH/Fab bi-specific molecules contain only one light chain in the Fab arm, co-expression of the VH-Fc knob with Fab-Fc hole should generate the proper VH/Fab bi-specific IgG . Using this strategy, we generated 4 different VH/Fab bi-specifics to explore combinations of targeting sites A and B with sites C and D; Bis1 is VH/Fab fusion, Bis2 is VH/Fab, Bis3 is VH/Fab, and Bis4 is VH/Fab. For each bispecific, the VH was cloned into the “knob” Fc, and the heavy chain of the Fab was cloned into the “hole” Fc.
All four bi-specific IgGs were successfully expressed and purified and shown by gel to be bi-specific, containing both a VH-Fc and Fab-Fc arm on each molecule . All 4 bi-specifics bound Secto with higher affinity than their parental mono-specific counterparts . In particular, Bis3 and Bis4, which both utilize Fab, bound Secto with highest affinity with KD,app of 395 pM and 127 pM, respectively. Reversing the orientation and immobilizing the bi-specific IgG on the biosensor and probing with soluble Secto did not significantly change the measured affinity . We then tested the neutralization potency of these VH/Fab bi-specifics using HIV-1-derived lentivirus pseudotyped with SARS-CoV-2 Spike to compare their potencies to the mono-specific bi-valent VH-Fc and IgGs. Spike pseudotypes were generated and used in neutralization assays with HEK293T cells expressing ACE2 on the surface using established vectors and protocols.VH-Fc A01 and VH-Fc B01 neutralized Spike pseudotypes with half-maximal inhibitory concentration of 2.86 nM and 2.01 nM , respectively. In contrast, we found that IgG C01 and IgG D01 did not neutralize pseudovirus at the concentrations tested . This is likely due to the fact that these two IgGs do not target the ACE2 binding site, and there are likely epitope and geometry-specific mechanisms that determine whether and to what extent non-blocking binders neutralize virus. Indeed, anti-Spike antibodies that do not compete with ACE2 binding, including CR3022, have been reported but with mixed and variable success in neutralizing SARS-CoV-2 depending on the specific antibody and its epitope.Next, we tested the neutralization profile of the bi-specific VH/Fab IgGs. To our surprise, we found that bi-specifics that utilize the non-neutralizing Fab D01 neutralized significantly more potently than the parental mono-specific binders. Bis3 and Bis4 neutralized with IC50s of 0.128 nM and 0.107 nM , respectively, which is ~20-fold more potent than the VH-Fc alone . This indicates that a bi-specific antibody, even if one of the arms is non-neutralizing, can show enhanced potency compared to the mono-specific counterparts.
In contrast, the bi-specific antibodies that utilized Fab C01 did not show such improved potency and neutralized with similar IC50s to the parental VH-Fc . This suggests that the enhanced potency provided by a non-neutralizing binder is epitope specific. Interestingly, we observe no significant correlation between IC50 and binding affinity against Secto of the bi-specifics and VH-Fcs . This suggests that an increase in neutralization potency of anti-Spike binders is not highly predictive from affinity alone, and that the specific mechanism and geometry of epitopes targeted by the bi-specific IgG scaffold likely play an important role in increasing potency. Additionally, we find that a cocktail of the monospecific antibodies does not improve potency , which suggests that a bi-specific IgG, where two epitopes are targeted by a single agent, has unique mechanisms and advantages. Lastly,vertical lettuce tower we examined the neutralization profile of the bi-specific IgGs and VH-Fc on authentic SARS-CoV-2 virus with Vero E6 cells as the host cell. Each binder was assayed for its ability to decrease the cytopathic effect of Vero E6 cells caused by SARS-CoV-2 infection. Consistent with the pseudovirus neutralization results, bi-specific IgGs Bis3 and Bis4 neutralized authentic SARS-CoV-2 significantly more potently than the VH-Fcs . The IC50s of VH-Fc A01 and VH-Fc B01 against authentic SARS-CoV-2 were 25-30 nM. These values are similar to a previously reported IC50 of VH-Fc B01 of 33.5 nM, which used qPCR to read out intracellular viral RNA.60 The neutralization IC50s of Bis3 and Bis4 against authentic SARS-CoV-2 were 1.00 nM and 1.19 nM , respectively. Interestingly, Bis1 and Bis2, which did not show a significant improvement over the VH-Fcs in the pseudovirus neutralization assays, were about 2.5 to 3-fold more potent than the VH-Fcs when assayed on authentic virus. Taken together, we find that bi-specific IgGs, particularly Bis3 and Bis4, are significantly more potent in neutralizing both pseudotyped and authentic SARS-CoV-2 virus than their mono-specific counterparts. Bis4, which uses the combination of VH B01 and Fab D01 was one of the most potent molecules we tested in this study. The epitope of VH B01 was determined previously by cryo-EM.In the absence of a high-resolution structure of Fab D01 bound to Spike, we utilized the structure of CR302264 as a surrogate to model how the two arms on Bis4 could engage Spike. We find that in the context of the same RBD, the VH and Fab bind at separate, non-overlapping epitopes . The CR3022 epitope is fully exposed only in the RBD “up” conformation, while the VH B01 epitope is largely accessible regardless of the RBD conformation. VH B01 and Fab CR3022 point away from each other at an angle of 133 degrees, with a distance of 134.5 Å between the C-termini of the VH and heavy chain of the Fab. Although the exact epitope and angle of engagement of Fab D01 may differ from CR3022, it appears challenging for both arms of Bis4 to engage the same RBD. However, it could be possible for the Bis4 VH arm to bind one RBD, and the Fab arm to bind a neighboring RBD in the context of the Spike trimer when one or more RBDs are in the “up” conformation .
We hypothesize that simultaneous engagement of two RBDs on Spike by Bis4 could explain its potent neutralization mechanism, although there could be other unexplored mechanisms at play. Determining the exact epitope and binding mechanism of Fab D01 would shed further light into how these bi-specific IgGs engage Spike and neutralize SARS-CoV-2. Here, we report the generation of bi-specific IgG antibodies against SARS-CoV-2 Spike that combine neutralizing and non-neutralizing binders against different epitopes on the Spike-RBD as a promising protein engineering strategy to rapidly improve the potency of antibodies against COVID-19. We show that certain combinations of these binders into a bi-specific IgG scaffold are significantly more potent in neutralizing pseudotyped and authentic SARS-CoV-2 virus than the mono-specific bi-valent counterparts and may enable efficacy increases not predicted by affinity alone. Additionally, we show that non-blocking, non-neutralizing epitopes can provide an unexpected benefit and boost the potency of molecules when combined into a bi-specific format with neutralizing epitopes. Combing neutralizing and non-neutralizing epitopes can be a useful approach to rescue binders that would have been deprioritized due to their lack of neutralization. Exploring the use of this strategy in parallel with other affinity maturation campaigns on some of the most potent antibodies reported against SARS-CoV-2 to date could enable the generation of even more potent neutralizing binders. This can decrease the effective dose necessary for therapeutic effect, thus lessen the burden on manufacturing capacity and enable the wider distribution of these treatments. In aggregate, we have produced binders against 4 distinct epitopes within the RBD of Spike. This is somewhat remarkable given the small size of the RBD . Binders to many epitopes on Spike have been reported,showing that this antigen is highly immunogenic both in vivo and in vitro. It will be interesting to map them relative to those we have found. Our results show that combinations via a KIH bi-specific scaffold is particularly useful to improve potency through avidity and multi-epitope targeting and could be less labor and time intensive than affinity maturation. Our study also highlights how different epitopes on Spike differ in their neutralization potency and their engineering potential. To what extent an antibody neutralizes SARS-CoV-2 is likely influenced by its binding mechanism, affinity, and scaffold, and is made more complex by the oligomeric and dynamic nature of the target antigen Spike. It is well established that the RBDs of Spike proteins of the coronavirus family are capable of adopting various conformations and that the trimer can undergo large conformational changes.These structural dynamics likely affect the accessibility and orientation of the different binding epitopes, which can then influence the potency of binders and how they behave when engineered into multi-specific modalities. This may help explain why bi-specifics that use Fab D01 are superior to bi-specifics that use Fab C01. Additionally, even binders that overlap in epitope, such as Fab D01 and CR3022, may differ in their properties. CR3022 was also non-neutralizing ,although another recent study contradicts this observation, and showed that CR3022 can neutralize SARS-CoV-2, possibly through the destabilization of the Spike trimer. Which combinations of binders will synergize and what antibody scaffolds are optimal for efficacy remain unclear, and provides a wide latitude for protein engineer to explore how these different factors affect antibody potency. A deeper biophysical understanding of Spike dynamics will also be important for rational engineering of potent biologics to this therapeutically important target. We believe these principles for design of bi-specific and bi-paratopic binders combining neutralizing and non-neutralizing binders can apply to other therapeutic targets even in the absence of high-resolution structures.