Nanobody repertoire generated against the spike protein of ancestral SARS-CoV-2 remains efficacious against the rapidly evolving virus

Reviewer #1 (Public Review):

Summary:
In this manuscript, Ketaren, Mast, Fridy et al. assessed the ability of a previously generated llama nanobody library (Mast, Fridy et al. 2021) to bind and neutralize SARS-CoV-2 delta and omicron variants. The authors identified multiple nanobodies that retain neutralizing and/or binding capacity against delta, BA.1 and BA.4/5. Nanobody epitope mapping on spike proteins using structural modeling revealed possible mechanisms of immune evasion by viral variants as well as mechanisms of cross-variant neutralization by nanobodies. The authors additionally identified two nanobody pairs involving non-neutralizing nanobodies that exhibited synergy in neutralization against the delta variant. These results enabled the refinement of target epitopes of the nanobody repertoire and the discovery of several pan-variant nanobodies for further preclinical development.

Strengths:
Overall, this study is well executed and provides a valuable framework for assessing the impact of emerging SARS-CoV-2 variants on nanobodies using a combination of in vitro biochemical and cellular assays as well as computational approaches. There are interesting insights generated from the epitope mapping analyses, which offer possible explanations for how delta and omicron variants escape nanobody responses, as well as how some nanobodies exhibit cross-variant neutralization capacity. These analyses laid out a clear path forward for optimizing these promising next-gen therapeutics, particularly in the face of rapidly emerging SARS-CoV-2 variants. This work will be of interest to researchers in the fields of antibody/nanobody engineering, SARS-CoV-2 therapeutics, and host-virus interaction.

Weaknesses:
A main weakness of the study is that the efficacy statement is not thoroughly supported. While the authors comprehensively characterized the neutralizing ability of nanobodies in vitro, there is no animal data involving mice or hamsters to demonstrate the real protective efficacy in vivo. Yet, in the title and throughout the manuscript, the authors repeatedly used phrases like "retains efficacy" or "remains efficacious" to describe the nanobodies' neutralization or binding capacities. This claim is not well supported by the data and underestimates the impact of variants on the nanobodies, especially the omicron sublineages. For example, the authors showed that S1-RBD-15 had a ~100-fold reduction in neutralization titer against Omicron, with an IC50 at around 1 uM. This is much higher than the IC50 value of a typical anti-ancestral RBD nanobody reported in the previous study (Mast, Fridy et al. 2021). In fact, the authors themselves ascribe nanobodies with an IC50 above 1 uM as weak neutralizers. And there were many in the range of 0.1-1 uM. Furthermore, many nanobodies selected for affinity measurement against BA.4/5 had no detectable binding. Without providing in vivo protection data or including monoclonal antibodies that are known to be efficacious against variants in the in vitro assays as a benchmark, it is difficult to evaluate the efficacy just with the IC50 values.

Reviewer #2 (Public Review):

Summary:
Interest in using nanobodies for therapeutic interventions in infectious diseases is growing due to their ability to bind hidden or cryptic epitopes that are inaccessible to conventional immunoglobulins. In the present study, the authors were posed to characterize nanobodies derived from the library produced earlier with the Wuhan strain of SARS-CoV-2, map their epitopes on SARS-CoV-2 spike protein, and demonstrate that some nanobodies retain binding and even neutralization against antigenically distant Variants of Concern (VOCs) that are currently circulating.

Strengths:
The authors demonstrate that some nanobodies - despite being obtained against the ancestral virus strain - retain high affinity binding to antigenically distant SARS-CoV-2 strains. This is despite the majority of the repertoire losing binding. Although limited to only two nanobody combinations, the demonstration of synergy in virus neutralization between nanobodies targeting different epitopes is compelling.

Weaknesses:
The authors imply that nanobodies that retain binding/neutralization of early Omicron sublineages will be active against currently circulating and future virus strains. Unfortunately, no reasoning for such a conclusion nor data supporting this prediction are provided.

Author Response

eLife assessment

This paper by Aitchison and colleagues describes nanobody neutralizing and binding activity against various SARS-CoV-2 variants of concern. The findings are important in that the described nanobodies may have broad therapeutic relevance against current and future variants of concern and may be able to avoid significant resistance. The claims are incomplete: while the study is well-executed and uses a nice balance of biochemical and cellular assays, the efficacy of the proposed nanobody library against VOCs is not completely supported as IC50 values appear to increase against newer variants and are higher than previously used therapeutic bNAbs, animal data showing in vivo efficacy is lacking, and protection against future possible variants is not proven.

This manuscript is a follow-up of our previous eLife manuscript “Highly synergistic combinations of nanobodies that target SARS-CoV-2 and are resistant to escape” https://elifesciences.org/articles/73027 where we described an “impressive collection of hundreds of new nanobodies binding SARS-CoV-2 spike by combining in vivo antibody affinity maturation and proteomics. [Editor’s evaluation]”. As a follow-up this submission extends the findings of our previous eLife publication and thus focuses on how our repertoire functions in the context of a rapidly evolving SARS-CoV-2 virus, relying on the established methodologies and approaches of the original paper. We explore how nanobody functions have been influenced by the emergence of SARS-CoV-2 variants containing extensive mutations in spike protein, which largely reduced the usefulness of therapeutic monoclonal antibody therapeutics. Our findings show that while some nanobodies lost efficacy in binding to and neutralizing these evolved spikes, a surprising number of nanobodies retained their binding and neutralization activity. This is an important finding, because these efficacious nanobodies target regions that appear rarely targetable by monoclonal antibodies. We also provide experimental validation of the importance of the interplay between binding and neutralization in synergy experiments, where even weakened binding still contributed to strongly enhancing the neutralization.

Reviewer #1 (Public Review):

Summary:

In this manuscript, Ketaren, Mast, Fridy et al. assessed the ability of a previously generated llama nanobody library (Mast, Fridy et al. 2021) to bind and neutralize SARS-CoV-2 delta and omicron variants. The authors identified multiple nanobodies that retain neutralizing and/or binding capacity against delta, BA.1 and BA.4/5. Nanobody epitope mapping on spike proteins using structural modeling revealed possible mechanisms of immune evasion by viral variants as well as mechanisms of cross-variant neutralization by nanobodies. The authors additionally identified two nanobody pairs involving non-neutralizing nanobodies that exhibited synergy in neutralization against the delta variant. These results enabled the refinement of target epitopes of the nanobody repertoire and the discovery of several pan-variant nanobodies for further preclinical development.

Strengths:

Overall, this study is well executed and provides a valuable framework for assessing the impact of emerging SARS-CoV-2 variants on nanobodies using a combination of in vitro biochemical and cellular assays as well as computational approaches. There are interesting insights generated from the epitope mapping analyses, which offer possible explanations for how delta and omicron variants escape nanobody responses, as well as how some nanobodies exhibit cross-variant neutralization capacity. These analyses laid out a clear path forward for optimizing these promising next-gen therapeutics, particularly in the face of rapidly emerging SARS-CoV-2 variants. This work will be of interest to researchers in the fields of antibody/nanobody engineering, SARS-CoV-2 therapeutics, and host-virus interaction.

Weaknesses:

A main weakness of the study is that the efficacy statement is not thoroughly supported. While the authors comprehensively characterized the neutralizing ability of nanobodies in vitro, there is no animal data involving mice or hamsters to demonstrate the real protective efficacy in vivo. Yet, in the title and throughout the manuscript, the authors repeatedly used phrases like "retains efficacy" or "remains efficacious" to describe the nanobodies' neutralization or binding capacities.

This claim is not well supported by the data and underestimates the impact of variants on the nanobodies, especially the omicron sublineages. For example, the authors showed that S1-RBD-15 had a ~100-fold reduction in neutralization titer against Omicron, with an IC50 at around 1 uM. This is much higher than the IC50 value of a typical anti-ancestral RBD nanobody reported in the previous study (Mast, Fridy et al. 2021). In fact, the authors themselves ascribe nanobodies with an IC50 above 1 uM as weak neutralizers. And there were many in the range of 0.1-1 uM.

Furthermore, many nanobodies selected for affinity measurement against BA.4/5 had no detectable binding.

Without providing in vivo protection data or including monoclonal antibodies that are known to be efficacious against variants in the in vitro assays as a benchmark, it is difficult to evaluate the efficacy just with the IC50 values.

We respectfully disagree with the reviewer on several aspects of this critique.

As to our use of the word efficacy - the quality of being successful in producing an intended result; effectiveness - we were specific to nanobody binding and in vitro neutralization of the variant spike proteins tested in the manuscript. Indeed, our manuscript made no claim of efficacy outside of this intended meaning. However, to prevent misinterpretation we will modify the final paragraph of our introduction to state explicitly that the nanobody repertoire retains efficacy in binding and neutralizing variants of spike. The final paragraph of the Introduction will include the following:

“Here, we demonstrate that a subset of our previously published repertoire of nanobodies, generated against spike from the ancestral SARS-CoV-2 virus (Mast, Fridy et al. 2021), retains binding and in vitro neutralization efficacy against circulating variants of concern (VoC), including omicron BA.4/BA.5.”

We agree that in vivo neutralization data would be an important complement to the in vitro binding and neutralization data. Experiments along these lines are ongoing, but are not considered part of a follow-up to our original paper where in vivo data were not included.

We disagree with the Reviewer that “This claim is not well supported by the data and underestimates the impact of variants on the nanobodies, especially the omicron sublineages.” As we specifically state: “In comparison, groups I, I/II, I/IV, V, VII, VIII and the anti-S2 nanobodies contained the majority of omicron BA.1 neutralizers, though here the neutralization potency of many nanobodies was decreased compared to wild-type. This decrease in neutralization potency largely correlates with the accumulation of omicron BA.1 specific mutations throughout the RBD, which likely alters the epitope-binding site of these nanobodies, weakening their interaction with BA.1 spike (Fig. 1B). (emphasis added)”

Naturally, we expected that some of our nanobodies would lose the ability to bind BA.4/BA.5. This enabled us to determine which areas on spike remained susceptible to our nanobodies. We show that 10/29 nanobodies tested retained binding to BA.4/5. We did not test our entire repertoire, just a subset was selected for. We stated the following:

“Of the nanobodies that neutralized both delta and omicron BA.1, representatives from each of the nanobody epitope groups were selected for SPR analysis, where S1 binders with mapped epitopes that neutralized one or both variants well, were prioritized.”

Reviewer #2 (Public Review):

Summary:

Interest in using nanobodies for therapeutic interventions in infectious diseases is growing due to their ability to bind hidden or cryptic epitopes that are inaccessible to conventional immunoglobulins. In the present study, the authors were posed (sic) to characterize nanobodies derived from the library produced earlier with the Wuhan strain of SARS-CoV-2, map their epitopes on SARS-CoV-2 spike protein, and demonstrate that some nanobodies retain binding and even neutralization against antigenically distant Variants of Concern (VOCs) that are currently circulating.

Strengths:

The authors demonstrate that some nanobodies - despite being obtained against the ancestral virus strain - retain high affinity binding to antigenically distant SARS-CoV-2 strains. This is despite the majority of the repertoire losing binding. Although limited to only two nanobody combinations, the demonstration of synergy in virus neutralization between nanobodies targeting different epitopes is compelling.

We thank the Reviewer for this positive summary of the strengths of our study. In our previous work, we applied stringent criteria for the down-selection of nanobodies based on their affinity and diversity, as elaborated on in https://elifesciences.org/articles/73027. The current dataset is a further judiciously curated subset, featuring 41 nanobodies chosen to represent and inform on the 10 structurally mapped epitope groups that we initially identified. This subset is but the tip of an iceberg. For each nanobody demonstrating high-affinity binding and neutralization, we possess multiple sequence variants, offering alternative avenues for investigation. Moreover, our repertoire has since been further elaborated by use of a yeast display library (Cross et al., 2023 JBC) providing additional nanobodies capable of targeting the same epitopes. Our findings presented here, thus serve as a heuristic, enabling us to distill the much larger repertoire into manageable and informative clusters of data. We will modify our manuscript to be more explicit of these facts.

Weaknesses:

The authors imply that nanobodies that retain binding/neutralization of early Omicron sublineages will be active against currently circulating and future virus strains. Unfortunately, no reasoning for such a conclusion nor data supporting this prediction are provided.

The nanobodies we propose to retain binding to current and emerging omicron sublineages at the time (Fig. 4) are those that still bind to omicron BA.1, BA.4/5. The structures of XBB and BQ.1 are not divergent enough from these aforementioned omicron sublineages in the regions we propose our nanobodies retain binding (Fig. 4) to result in loss of binding. Thus, we hypothesize that the epitopes where these nanobodies bind or are predicted to bind (outlined in black (Fig. 4)), represent regions on spike vulnerable to nanobody intervention. Importantly, we also now have further experimental data to support our predictions that these nanobodies in Fig. 4 will retain binding (see plot in Author response image 1). We will provide additional data and complements to key figures to help illustrate this in the revised manuscript.

Author response image 1.