De novo-designed minibinders expand the synthetic biology sensing repertoire

Reviewer #1 (Public Review):

Summary:

The authors want to explore how much two known minibinder protein domains against the Spike protein of SARS-CoV-2 can function as a binding domain of 2 sets of synthetic receptors (SNIPR and CAR); the authors also want to know how some modifications of the linkers of these new receptors affect their activation profile.

Major strengths and weaknesses of the methods and results:

- Strengths include: analysis of synthetic receptor function for 2 classes of synthetic receptors, with robust and appropriate assays for both kinds of receptors. The modifications of the linkers are also interesting and the types of modifications that are often used in the field.

- Weaknesses include: none of the data analysis provides statistical interpretation of the results (that I could find). One dataset is confusing: Figures 5A and C, are said to be the same assay with the same constructs, but the results are 30% in A, and 70% in C.

An appraisal of whether the authors achieved their aims, and whether the results support their conclusions:

Given the open-ended nature of the goal (implicit in it being an exploration), it is hard to say if the authors have reached their aims; they have done an exploration for sure; is it big enough an exploration? This reviewer is not sure.

The results are extremely clearly presented, both in the figures and in the text, both for the methods and the results. The claims put forward (with limited exceptions see below) are very solidly supported by the presented data.

A discussion of the likely impact of the work on the field, and the utility of the methods and data to the community:

The work may stimulate others to consider minibinders as potential binding domains for synthetic receptors. The modifications that are presented although not novel, do provide a starting point for larger-scale analysis.

It is not clear how much this is generalizable to other binders (the authors don't make such claims though). The claims are very focused on the tested modifications, and the 2 receptors and minibinder used, a scope that I would define as narrow; the take-home message if one wants to try it with other minibinders or other receptors seems to be: test a few things, and your results may surprise you.

Any additional context you think would help readers interpret or understand the significance of the work:

We are at the infancy stage of synthetic receptors optimization and next-generation derivation; there is a dearth of systematic studies, as most focus is on developing a few ones that work. This work is an interesting attempt to catalyze more research with these new minibinders. Will it be picked up based on this? Not sure.

Reviewer #2 (Public Review):

Summary:

Weinberg et al. show that spike LCB minibinders can be used as the extracellular domain for SynNotch, SNIPR, and CAR. They evaluated their designs against cells expressing the target proteins and live virus.

Strengths:

This is a good fundamental demonstration of alternative use of the minibinder. The results are unsurprising but robust and solid in most cases.

Weaknesses:

The manuscript would benefit from better descriptions of the study's novelty. Given that LCB previously worked in SynNotch, what unexpected finding was uncovered by this study? It is well known that the extracellular domain of CAR is amendable to different types of binding domains (e.g., scFv, nanobody, DARPin, natural ligands). So, it is not surprising that a minibinder also works with CAR. We don't know if the minibinders are more or less likely to be compatible with CAR or SNIPR.

The demonstrations are all done using just 1 minibinder. It is hard to conclude that minibinders, as a unique class of protein binders, are generalizable in different contexts. All it can conclude is that this specific Spike minibinder can be used in synNotch, SNIPR, and CAR. The LCB3 minibinder seems to be much weaker.

The sensing of live viruses is interesting, but the output is very weak. It is difficult to imagine a utility for such a weak response.

Author response:

We thank the reviewers for their attention to our study and for their fair and reasonable assessment of the strengths and weaknesses of our work. We believe the reviewers adequately captured both the potential implications of our work as well as its major current limitations. As both reviewers noted, we believe the work presented in this manuscript is an exciting first step in adapting minibinders as antigen sensors for synthetic receptors but many questions remain before these new tools can be widely adopted. We hope that this work will catalyze others to try minibinders as potential antigen sensors when developing novel synthetic receptors, and we hope that future work will more thoroughly test a wide range of linkers to better optimize antigen sensor function across synthetic receptors.

In our future work, we intend to evaluate a greater diversity of minibinders across different relevant therapeutic targets. We are working to test both existing minibinders as well as generate novel minibinders using deep-learning-based de novo protein design methods. We further hope to explore additional linker modifications, especially focusing on modifications that will allow minibinder coupled-synthetic receptors to escape the glycocalyx of engineered cells. We hope to share findings on these topics in either an update to this manuscript or in future manuscripts, depending on the results of our studies in progress.

Finally, reviewers noted a mismatch in the data displayed in Figure 5A and 5C, whereby LCB-CAR-expressing cells induced higher lysis in Figure 5C than in Figure 5A. This is due to figure 5C showing only 24 hours of incubation between effector and target cells, as opposed to the 72 hours of incubation that is quantitated in 5A. These mismatched timepoints were selected because linker-dependent differences in lysis were most readily apparent at 24 hours and were negligible at 72 hours. The full-time course of lysis for this experiment can be seen in Supplemental Figure 2D.