From the journal RSC Chemical Biology Peer review history

12-Sep-2021

Dear Dr Cheloha:

Manuscript ID: CB-ART-05-2021-000118
TITLE: Activation of a G protein-coupled receptor through indirect antibody-mediated tethering of ligands

Thank you for your submission to RSC Chemical Biology, published by the Royal Society of Chemistry. I sent your manuscript to reviewers and I have now received their reports which are copied below. My apologies again that it took so long.

After careful evaluation of your manuscript and the reviewers’ reports, I will be pleased to accept your manuscript for publication after revisions.

Please revise your manuscript to fully address the reviewers’ comments. When you submit your revised manuscript please include a point by point response to the reviewers’ comments and highlight the changes you have made. Full details of the files you need to submit are listed at the end of this email.

Please submit your revised manuscript as soon as possible using this link :

*** PLEASE NOTE: This is a two-step process. After clicking on the link, you will be directed to a webpage to confirm. ***

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Alternatively, you can login to your account (https://mc.manuscriptcentral.com/rsccb) where you will need your case-sensitive USER ID and password.

You should submit your revised manuscript as soon as possible; please note you will receive a series of automatic reminders. If your revisions will take a significant length of time, please contact me. If I do not hear from you, I may withdraw your manuscript from consideration and you will have to resubmit. Any resubmission will receive a new submission date.

Supporting our community through Covid-19
While our publishing services are running as usual, we also know that this is a very challenging time for everyone, for many different reasons. If any aspect of the publishing process is worrying you – for example you think you may struggle to meet a pre-determined deadline – please let us know, and we will work out an answer together.

RSC Chemical Biology strongly encourages authors of research articles to include an ‘Author contributions’ section in their manuscript, for publication in the final article. This should appear immediately above the ‘Conflict of interest’ and ‘Acknowledgement’ sections. I strongly recommend you use CRediT (the Contributor Roles Taxonomy from CASRAI, https://casrai.org/credit/) for standardised contribution descriptions. All authors should have agreed to their individual contributions ahead of submission and these should accurately reflect contributions to the work. Please refer to our general author guidelines http://www.rsc.org/journals-books-databases/journal-authors-reviewers/author-responsibilities/ for more information.

The Royal Society of Chemistry requires all submitting authors to provide their ORCID iD when they submit a revised manuscript. This is quick and easy to do as part of the revised manuscript submission process. We will publish this information with the article, and you may choose to have your ORCID record updated automatically with details of the publication.

Please also encourage your co-authors to sign up for their own ORCID account and associate it with their account on our manuscript submission system. For further information see: https://www.rsc.org/journals-books-databases/journal-authors-reviewers/processes-policies/#attribution-id

Please note: to support increased transparency, RSC Chemical Biology offers authors the option of transparent peer review. If authors choose this option, the reviewers’ comments, authors’ response and editor’s decision letter for all versions of the manuscript are published alongside the article. Reviewers remain anonymous unless they choose to sign their report. We will ask you to confirm whether you would like to take up this option at the revision stages.

I look forward to receiving your revised manuscript.

Yours sincerely,
Dr Andrea Rentmeister

************

Reviewer 1

Review of Cheloha et al. 2021 RSC Chemical Biology Activation of a G protein-coupled receptor through indirect antibody-mediated tethering of ligands

Cheloha and colleagues report on the use of single domain antibody fragments (nanobodies/VHH) conjugated with parathyroid hormone receptor 1 (PTHR1) ligand fragments to indirectly target PTHR1 in living cells. Rather than binding the PTHR1 directly, the nanobodies were targeted to other membrane proteins, including MHC Class I or integrins, or against murine Igs. They combined these in non-natural fusions with PHTR ligand fragments (both 1-11 and 1-34) called CLAMPs. This approach was taken in an effort to expand the potential for antibody-conjugated tools for GPCR research, as relatively few GPCRs have suitable antibodies targeted against them, compared to other membrane proteins. They first showed CLAMPs could indeed activate PTHR1, despite binding other membrane proteins, albeit as weak agonists. Indirect targeting of PTHR1 with a murine Ig-specific nanobody-ligand conjugate and a bridging IgG greatly increased the ability for the tethered nanobody to activate the receptor. To expand the potential use for these conjugated nanobodies in vivo, the authors utilised the same approach but with a nanobody targeted against rabbit IgG. This would enable their use in murine models without the nanobody binding circulating murine Igs in the bloodstream. This rabbit Ig-targeted nanobody also displayed agonist activity at the PTHR1. Finally, through co-culture methods with adherent and suspension cells, the authors demonstrate it possible for CLAMPs to activate their target receptor intercellularly.
Overall, this body of work represents an expansion of the potential toolkit through which GPCRs can be interrogated with biologics. It removes the issue with receptor-specific nanobodies (for which there aren’t many), instead using well-characterised nanobodies against other membrane protein targets or Igs. However, the target protein must be chosen carefully, as it was not always possible for differentially targeted CLAMPs to activate the PTHR1, e.g. those via antibodies against CD63. In showing CLAMPs can activate their receptor through indirect targeting, this study signifies an exciting advance in the ability to study GPCR signalling with antibodies.

This paper is technically sound with clearly described methods and aims. I do have a few minor comments to the authors:
- The discussion around the differences between the results in fig. 2e could be expanded upon, regarding the small response seen with VHHα5β1 vs VHHMHC-I. Could the authors suggest a reason as to why this may occur? Could subcellular localisation be a factor here?

- I feel supplementary figure 3 should be included in the main text. The confirmation that rabbit Ig-targeted CLAMPs work just as well as their murine counterparts is important for the potential in vivo applications in murine models and thus justifies inclusion in the main text.

- Page 17, first paragraph, 4th line: ‘grows’ should be ‘grow’ as it refers to the HEK293 cells.

- Why have the data in fig. 5b not been fitted to sigmoidal dose-response models?

Reviewer 2

This is an extremely interesting study investigating ligand-conjugated nanobodies as an approach to deliver ligands to a specific target of interest. The authors have explored three different strategies that build on their previous work. Of particular interest to this reviewer is the strategies highlighted in Figure 1b and 1d, where the nanobody portion of the conjugated ligand construct is targeted at neighbouring proteins. It is likely that the failure of strategy 1b is related to linker length and I wonder whether the authors have investigated different lengths of tether in any detail? The success of the monoclonal antibody strategy depicted in Figure 1d would suggest that this is probably the case. It would also seem important to check whether the VHHb2m-PTH11 conjugate lacked both the ability to bind to the PTHR1 receptor as well as to elicit a functional agonist response. For example, it could be tested in antagonist mode against the standard PTH(1-34) full agonist.

My major concern with the manuscript as presented is the lack of proper statistical analysis of the data generated. Although mean and SDs are shown there is no indication whether the changes are significant or not. For example, In Figure 3a there is a large error associated with the data for PTH(1-11) using nanobody VHHmig and an alpha GFP antibody. Is this significantly different from basal? If not then one would come to a very different conclusion. I think statistical analysis of the effects presented in the Figures should be presented with p values and the tests used.

REVIEWER 1:
COMMENT: Overall, this body of work represents an expansion of the potential toolkit through which GPCRs can be interrogated with biologics. It removes the issue with receptor-specific nanobodies (for which there aren’t many), instead using well-characterised nanobodies against other membrane protein targets or Igs. However, the target protein must be chosen carefully, as it was not always possible for differentially targeted CLAMPs to activate the PTHR1, e.g. those via antibodies against CD63. In showing CLAMPs can activate their receptor through indirect targeting, this study signifies an exciting advance in the ability to study GPCR signalling with antibodies.

RESPONSE: We appreciate the comments from reviewer 1.

COMMENT: This paper is technically sound with clearly described methods and aims. I do have a few minor comments to the authors:
- The discussion around the differences between the results in fig. 2e could be expanded upon, regarding the small response seen with VHHα5β1 vs VHHMHC-I. Could the authors suggest a reason as to why this may occur? Could subcellular localisation be a factor here?

RESPONSE: We do not know the basis for this surprising finding, so we can only speculate. At the end of the second paragraph of the “results and discussion” section we have added text reading “The origin of this difference is unknown, but it may relate to the larger size and expanded flexibility of the extracellular portions of α5β1 relative to β2-microglobulin as a more compact subunit of MHC-I. Another possibility is that PTHR1 may localize to membrane domains closer to α5β1 relative to the β2-microglobulin-MHC-I complex.”

COMMENT: - I feel supplementary figure 3 should be included in the main text. The confirmation that rabbit Ig-targeted CLAMPs work just as well as their murine counterparts is important for the potential in vivo applications in murine models and thus justifies inclusion in the main text.

RESPONSE: We have merged supplementary figure 3 with main text figure 3 (rabbit Ig data now found in Figure 3e-f)

COMMENT: - Page 17, first paragraph, 4th line: ‘grows’ should be ‘grow’ as it refers to the HEK293 cells.
RESPONSE: We have corrected this error.

COMMENT: - Why have the data in fig. 5b not been fitted to sigmoidal dose-response models?
RESPONSE: We have now fitted the data in Fig. 5b to a sigmoidal dose-response model.


Referee: 2

Comments to the Author
COMMENT: This is an extremely interesting study investigating ligand-conjugated nanobodies as an approach to deliver ligands to a specific target of interest. The authors have explored three different strategies that build on their previous work. Of particular interest to this reviewer is the strategies highlighted in Figure 1b and 1d, where the nanobody portion of the conjugated ligand construct is targeted at neighbouring proteins. It is likely that the failure of strategy 1b is related to linker length and I wonder whether the authors have investigated different lengths of tether in any detail? The success of the monoclonal antibody strategy depicted in Figure 1d would suggest that this is probably the case.
RESPONSE: We have previously evaluated the impact of the length of the linker between receptor agonist and a nanobody that binds to that same receptor (Cheloha et al Nature Communications 2020). We found that insertion of a modestly sized polyethylene glycol linker (PEG4) had a small impact on receptor agonist activity. As the reviewer notes, the strategy that relies on linking via a monoclonal antibody (figure 1d) was more successful. It has the added advantage of relying on widely commercially available monoclonal antibodies. We have therefore added text to the results and discussion section, as follows: “In this context, the use of a longer flexible linker between the receptor agonist and nanobody may improve activation efficacy. A conjugate consisting of a nanobody and ligand that both target the same receptor was relatively insensitive to inclusion of a short polyethylene glycol linker2.”

COMMENT: It would also seem important to check whether the VHHb2m-PTH11 conjugate lacked both the ability to bind to the PTHR1 receptor as well as to elicit a functional agonist response. For example, it could be tested in antagonist mode against the standard PTH(1-34) full agonist.
RESPONSE:PTH1-11 and variant peptides compete for PTH1-34 binding only at very high concentrations (>10 uM) (Shimizu et al. J. Biol. Chem. 2001, PMID 11604398). Such concentrations are higher than the maximal concentrations achievable for these nanobody-PTH conjugates. Further, the PTH1-11 variant used in this study strictly acts as a fully efficacious agonist (see also Cheloha et al Nature Communications 2020). We therefore consider it highly unlikely that nanobody-PTH1-11 conjugates would show any antagonist activity (in the absence of agonist activity).

COMMENT: My major concern with the manuscript as presented is the lack of proper statistical analysis of the data generated. Although mean and SDs are shown there is no indication whether the changes are significant or not. For example, In Figure 3a there is a large error associated with the data for PTH(1-11) using nanobody VHHmig and an alpha GFP antibody. Is this significantly different from basal? If not then one would come to a very different conclusion. I think statistical analysis of the effects presented in the Figures should be presented with p values and the tests used.
RESPONSE: We have now included statistical analyses to compare data sets (See Figures 3-4) along with a description of the tests used.

27-Sep-2021

Dear Dr Cheloha:

Manuscript ID: CB-ART-05-2021-000118.R1
TITLE: Activation of a G protein-coupled receptor through indirect antibody-mediated tethering of ligands

Thank you for your submission to RSC Chemical Biology, published by the Royal Society of Chemistry. I sent your manuscript to reviewers and I have now received their reports which are copied below.

After careful evaluation of your manuscript and the reviewers’ reports, I will be pleased to accept your manuscript for publication after revisions.

Please revise your manuscript to fully address the reviewers’ comments. When you submit your revised manuscript please include a point by point response to the reviewers’ comments and highlight the changes you have made. Full details of the files you need to submit are listed at the end of this email.

Please submit your revised manuscript as soon as possible using this link :

*** PLEASE NOTE: This is a two-step process. After clicking on the link, you will be directed to a webpage to confirm. ***

https://mc.manuscriptcentral.com/rsccb?link_removed

(This link goes straight to your account, without the need to log in to the system. For your account security you should not share this link with others.)

Alternatively, you can login to your account (https://mc.manuscriptcentral.com/rsccb) where you will need your case-sensitive USER ID and password.

You should submit your revised manuscript as soon as possible; please note you will receive a series of automatic reminders. If your revisions will take a significant length of time, please contact me. If I do not hear from you, I may withdraw your manuscript from consideration and you will have to resubmit. Any resubmission will receive a new submission date.

Supporting our community through Covid-19
While our publishing services are running as usual, we also know that this is a very challenging time for everyone, for many different reasons. If any aspect of the publishing process is worrying you – for example you think you may struggle to meet a pre-determined deadline – please let us know, and we will work out an answer together.

RSC Chemical Biology strongly encourages authors of research articles to include an ‘Author contributions’ section in their manuscript, for publication in the final article. This should appear immediately above the ‘Conflict of interest’ and ‘Acknowledgement’ sections. I strongly recommend you use CRediT (the Contributor Roles Taxonomy from CASRAI, https://casrai.org/credit/) for standardised contribution descriptions. All authors should have agreed to their individual contributions ahead of submission and these should accurately reflect contributions to the work. Please refer to our general author guidelines http://www.rsc.org/journals-books-databases/journal-authors-reviewers/author-responsibilities/ for more information.

The Royal Society of Chemistry requires all submitting authors to provide their ORCID iD when they submit a revised manuscript. This is quick and easy to do as part of the revised manuscript submission process. We will publish this information with the article, and you may choose to have your ORCID record updated automatically with details of the publication.

Please also encourage your co-authors to sign up for their own ORCID account and associate it with their account on our manuscript submission system. For further information see: https://www.rsc.org/journals-books-databases/journal-authors-reviewers/processes-policies/#attribution-id

Please note: to support increased transparency, RSC Chemical Biology offers authors the option of transparent peer review. If authors choose this option, the reviewers’ comments, authors’ response and editor’s decision letter for all versions of the manuscript are published alongside the article. Reviewers remain anonymous unless they choose to sign their report. We will ask you to confirm whether you would like to take up this option at the revision stages.

I look forward to receiving your revised manuscript.

Yours sincerely,
Dr Andrea Rentmeister

************

Reviewer 2

I think the manuscript had been improved by the additional of statistical analysis. However, the test used seems inappropriate. Unpaired t tests have been used in situations where the same data set has been compared with different groups. One-way ANOVA would be more appropriate to deal with these multiple comparisons, and a suitable post-hoc test can be used to test differences between specific combinations. I raise this since I think some of the differences will turn out to be non-significant once the impact of re-use of data-sets has been taken into consideration. ANOVA and appropriate post-hoc tests for multiple comparisons are available in Prism.

We are very pleased the manuscript has been deemed suitable for publication. Thank you for your handling of our submission.

We have changed the statistical test used to analyze data and this change is incorporated in the new version of the manuscript. Conclusions regarding the statistical significance of differences using this new test is unchanged (with one exception, Figure 3f now has one additional statistically significant difference).

30-Sep-2021

Dear Dr Cheloha:

Manuscript ID: CB-ART-05-2021-000118.R2
TITLE: Activation of a G protein-coupled receptor through indirect antibody-mediated tethering of ligands

Thank you for your submission to RSC Chemical Biology, published by the Royal Society of Chemistry. I sent your manuscript to reviewers and I have now received their reports which are copied below. Could it be the case that you uploaded the old version of the manuscript? Please double-check and re-submit the revised version.

Please revise your manuscript to fully address the reviewers’ comments. When you submit your revised manuscript please include a point by point response to the reviewers’ comments and highlight the changes you have made. Full details of the files you need to submit are listed at the end of this email.

Please submit your revised manuscript as soon as possible using this link :

*** PLEASE NOTE: This is a two-step process. After clicking on the link, you will be directed to a webpage to confirm. ***

https://mc.manuscriptcentral.com/rsccb?link_removed

(This link goes straight to your account, without the need to log in to the system. For your account security you should not share this link with others.)

Alternatively, you can login to your account (https://mc.manuscriptcentral.com/rsccb) where you will need your case-sensitive USER ID and password.

You should submit your revised manuscript as soon as possible; please note you will receive a series of automatic reminders. If your revisions will take a significant length of time, please contact me. If I do not hear from you, I may withdraw your manuscript from consideration and you will have to resubmit. Any resubmission will receive a new submission date.

Supporting our community through Covid-19
While our publishing services are running as usual, we also know that this is a very challenging time for everyone, for many different reasons. If any aspect of the publishing process is worrying you – for example you think you may struggle to meet a pre-determined deadline – please let us know, and we will work out an answer together.

RSC Chemical Biology strongly encourages authors of research articles to include an ‘Author contributions’ section in their manuscript, for publication in the final article. This should appear immediately above the ‘Conflict of interest’ and ‘Acknowledgement’ sections. I strongly recommend you use CRediT (the Contributor Roles Taxonomy from CASRAI, https://casrai.org/credit/) for standardised contribution descriptions. All authors should have agreed to their individual contributions ahead of submission and these should accurately reflect contributions to the work. Please refer to our general author guidelines http://www.rsc.org/journals-books-databases/journal-authors-reviewers/author-responsibilities/ for more information.

The Royal Society of Chemistry requires all submitting authors to provide their ORCID iD when they submit a revised manuscript. This is quick and easy to do as part of the revised manuscript submission process. We will publish this information with the article, and you may choose to have your ORCID record updated automatically with details of the publication.

Please also encourage your co-authors to sign up for their own ORCID account and associate it with their account on our manuscript submission system. For further information see: https://www.rsc.org/journals-books-databases/journal-authors-reviewers/processes-policies/#attribution-id

Please note: to support increased transparency, RSC Chemical Biology offers authors the option of transparent peer review. If authors choose this option, the reviewers’ comments, authors’ response and editor’s decision letter for all versions of the manuscript are published alongside the article. Reviewers remain anonymous unless they choose to sign their report. We will ask you to confirm whether you would like to take up this option at the revision stages.

I look forward to receiving your revised manuscript.

Yours sincerely,
Dr Andrea Rentmeister

************

Reviewer 2

As far as I can see the MS is identical to the previous one. Unpaired t-tests are still used and Figure 3F is identical (despite the comment from the authors) - I suspect they have uploaded the previous version.

Thank you for this response. I have made sure that the version of the manuscript uploaded with this submission is one with the ANOVA statistical test included. For reference I have also uploaded a version of the manuscript (marked changes) with all changes highlighted with red font. This version also includes the old version of Figure 3 and new version of Figure 3 for comparison. One of the comparisons that was non significant in the old version is now significant using the new statistical method.

01-Oct-2021

Dear Dr Cheloha:

Manuscript ID: CB-ART-05-2021-000118.R3
TITLE: Activation of a G protein-coupled receptor through indirect antibody-mediated tethering of ligands

Thank you for submitting your revised manuscript to RSC Chemical Biology. After considering the changes you have made, I am pleased to accept your manuscript for publication in its current form. I have copied any final comments from the reviewer(s) below.

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With best wishes,

Dr Andrea Rentmeister

Reviewer 2

The authors have dealt with the points I raised previously.