De novo-designed minibinders expand the synthetic biology sensing repertoire
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Curated by eLife
eLife Assessment
This study presents a useful investigation of the use of small, de novo-designed protein binding domains (mini-binders) against the Spike protein of SARS-CoV-2 and EGFR, as ligand binding domains on two classes of synthetic receptors, second-generation synNotch (SNIPR) and CAR. The methods and evidence supporting the focused claims are solid. This work will be of interest to synthetic biologists and cell engineers as a starting point to map out the rules for receptor engineering based on mini-binders and ultimately to advance them in biomedical applications.
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Abstract
Synthetic and chimeric receptors capable of recognizing and responding to user-defined antigens have enabled “smart” therapeutics based on engineered cells. These cell engineering tools depend on antigen sensors which are most often derived from antibodies. Advances in the de novo design of proteins have enabled the design of protein binders with the potential to target epitopes with unique properties and faster production timelines compared to antibodies. Building upon our previous work combining a de novo -designed minibinder of the Spike protein of SARS-CoV-2 with the synthetic receptor synNotch (SARSNotch), we investigated whether minibinders can be readily adapted to a diversity of cell engineering tools. We show that the Spike minibinder LCB1 easily generalizes to a next-generation proteolytic receptor SNIPR that performs similarly to our previously reported SARSNotch. LCB1-SNIPR successfully enables the detection of live SARS-CoV-2, an improvement over SARSNotch which can only detect cell-expressed Spike. To test the generalizability of minibinders to diverse applications, we tested LCB1 as an antigen sensor for a chimeric antigen receptor (CAR). LCB1-CAR enabled CD8+ T cells to cytotoxically target Spike-expressing cells. We further demonstrate that two other minibinders directed against the clinically relevant epidermal growth factor receptor are able to drive CAR-dependent cytotoxicity with efficacy similar to or better than an existing antibody-based CAR. Our findings suggest that minibinders represent a novel class of antigen sensors that have the potential to dramatically expand the sensing repertoire of cell engineering tools.
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eLife Assessment
This study presents a useful investigation of the use of small, de novo-designed protein binding domains (mini-binders) against the Spike protein of SARS-CoV-2 and EGFR, as ligand binding domains on two classes of synthetic receptors, second-generation synNotch (SNIPR) and CAR. The methods and evidence supporting the focused claims are solid. This work will be of interest to synthetic biologists and cell engineers as a starting point to map out the rules for receptor engineering based on mini-binders and ultimately to advance them in biomedical applications.
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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 can 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 …
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 can 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.
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Author response:
The following is the authors’ response to the original reviews.
In our initial submission, reviewers highlighted that the major limitations of our study were related to both the number of minibinders tested as well as the number of optimizations we evaluated for improving minibinder function. In this revision, we have focused on expanding the minibinders tested. To do so, we selected two previously published minibinders against the epidermal growth factor receptor (EGFR). Selection of EGFR as a target enabled us to evaluate two minibinders that bind at different sites, unlike the previously evaluated binders LCB1 and LCB3 which both bind the same interface on SARS-CoV-2 Spike. Further, using EGFR as a target enabled us to qualitatively compare the efficacy of minibinder-coupled chimeric antigen receptors against an …
Author response:
The following is the authors’ response to the original reviews.
In our initial submission, reviewers highlighted that the major limitations of our study were related to both the number of minibinders tested as well as the number of optimizations we evaluated for improving minibinder function. In this revision, we have focused on expanding the minibinders tested. To do so, we selected two previously published minibinders against the epidermal growth factor receptor (EGFR). Selection of EGFR as a target enabled us to evaluate two minibinders that bind at different sites, unlike the previously evaluated binders LCB1 and LCB3 which both bind the same interface on SARS-CoV-2 Spike. Further, using EGFR as a target enabled us to qualitatively compare the efficacy of minibinder-coupled chimeric antigen receptors against an existing anti-EGFR CAR. We believe the results here demonstrate broader generalizability of our approach across binding sites, targets, and minibinders. We hope this addition is sufficient to convince future would-be users of these tools to attempt synthetic receptor engineering using minibinders against their protein of choice.
Reviewers made comments about the presentation of flow data and the use of statistics throughout the manuscript. We did not modify how flow data are presented as the density plots we used are common throughout the field. We have opted to not include statistics – we believe that in the case of most of the experiments we show, our findings are obvious. In cases where statistics would be helpful for discerning whether subtle effects are real – for example, comparing the linker-based optimizations or comparing the anti-EGFR CARs – we believe that other experimental factors like construct expression are sufficient confounds that even in the presence of statistically significant effects we would be leading readers astray to make such claims about our data. As such, we have sought to limit the claims we make and hope that reviewers and audience agree we do not over interpret our data without statistical support.
On more minor points, both reviewers addressed the differences in Figure 5A and 5C, which we addressed in our figure legend and in the previous response to reviews is the result of these data originating from different time points of the same assay. Reviewer #2 believed we should be more staid in our comments about linker optimality, which we have addressed by changing the referenced line in the discussion. Otherwise, we have made no modifications to figures or text beyond the addition of new data.
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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 …
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.
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eLife assessment
This study presents a useful investigation to test de novo-designed mini binders against the Spike protein of SARS-CoV-2 within two classes of synthetic receptors (SNIPRs and CARs). The methods and evidence supporting the focused claims are very solid, although the small-scale nature of the investigation (number of modifications, number of minibinders, etc.) makes it difficult to determine how generalizable these results and potential design principles are. This work will be of interest to synthetic biologists and cell engineers as a starting point for systematic, larger-scale analysis and optimization of synthetic receptor designs for cellular therapy and other applications.
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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: …
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.
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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 …
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.
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