From the journal RSC Chemical Biology Peer review history

24-Feb-2021

Dear Dr Ploegh:

Manuscript ID: CB-REV-02-2021-000023
TITLE: Nanobodies: Powerful Tools For In Vivo, Non-Invasive, Imaging

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.

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I look forward to receiving your revised manuscript.

Yours sincerely,
Dr Andrea Rentmeister

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

Reviewer 1

In this manuscript, the authors provide an overview of the use of Nanobodies for in vivo nuclear and optical imaging applications. The review is well-written, has a logical build-up and is comprehensive. Except for a few very minor comments (typo's), I recommend that the manuscript should be accepted as it is for publication.


Pg 5: As more relevant references for the deep tissue penetration of nanobodies, the authors could refer to the following papers:
- Hernández, 2019, Mol Imaging Biol
- Debie, 2020, J Control Release

Pg 7: "This is often accompanied by uptake of the imaging agent in the kidneys and the liver."
Normally, hepatic clearance is not expected with Nanobodies, so I propose to remove the last part of the sentence. Liver uptake is only seen in case of constitutive expression of the target in liver tissue, or in case the imaging label negatively affects the nanobody's biodistribution.

Pg 13: 99mTC --> 99mTc

Pg 15: "Despite a slightly higher accumulation in the bladder"
--> Accumulation in the bladder is not relevant since this highly depends on whether the mouse has urinated just before being subjected to an imaging scan or not.

Table 3: The IRDye800CW-labeled anti-CEA nanobody misses in this table as image-guided surgery agent. (Lwin, 2020, Surgery)

Reviewer 2

In this review article, Ploegh and co-workers provide an overview on imaging applications of nanobodies in model organisms like mice and in human patients. The nanobodies are used as specific targeting molecules for the actual label, which is covalently attached. Nanobodies exhibit important advantages over other targeting units, in particular full-length antibodies or other antibody fragments due to their unique properties with respect to size, tissue penetration, rapid clearance, ease of recombinant protein production and protein stability.

This field is rapidly expanding and of increasing importance. The multidisciplinary approach can be viewed from different angles and I was a bit surprised of the focus of this review. For a journal like RSC Chemical Biology, I was expecting a focus on the chemical conjugation and ligation strategies to obtain the labeled nanobodies. However, the emphasis of the present manuscript is clearly on the biomedically relevant issues. In particular, the imaging modalities (PET, SPECT, etc.), the specific nanobodies used (anti-EGFR, anti CD8+ T-cells, etc.) and the medical questions (imaging of tumor cells, specific tumor environment, etc.) are discussed in the most detail. Comprehensive Tables list relevant nanobody molecules and provide a good overview over the literature. All these issues are highly relevant and nicely compiled in this review, however, they may not represent the typical scope of a journal focused on Chemical Biology.
This being said, I found the review excellent for its intended purpose. Not only does it probably fill a void much more than a protein-modification-centered view. The latter has been covered recently with many other reviews. I think the present review can become an important reference on the truly biomedical perspective of using nanobodies for in vivo imaging. Given the impressive importance of the modified nanobodies as key protein reagents for these applications, this can be seen as an instructive example how far Chemical Biology can reach into translational research and therefore be of more general interest to the readership of RSC Chem Biol.


Below is a list of minor points that should be addressed before publication:
- Page, please include a reference for 99mTc-labeling of a His6-tag.
- Figure 2 A and C, shown are various widely used chelators and dyes. However, since these are discussed in the context of their covalent attachment to nanobodies, please provide at least some of the structures as representative reagents for the bioconjugation reactions, for example as a maleimide reagent etc.. This information is useful for the reader to see the typical reagents and to get a feeling about which parts of the molecules can be modified for the attachment.
- Figure 3, the differences between the solid, dashed and dotted lines are very hard to appreciate
- Please re-iterate on the renal clearance issue associated with nanobodies. On the one hand, the advantages are highlighted, i.e., yielding a high signal-to-background ratio for imaging already 1 h after nanobody administration. On the other hand, renal uptake can be problematic for several reasons and can be modulated (by sequence, by PEG). However, would alleviating the latter problem mean that one loses again the advantage of the fast clearance? Can sequences and PEG also influence the clearance from the kidney, such that both could be accomplished, fast clearance from the circulation and fast clearance from the kidney?
- Page 10/11, include reference to Figure 4
- A few typos, e.g., biorthogonal > bioorthogonal, 650nm > 650 nm

This text has been copied from the PDF response to reviewers and does not include any figures, images or special characters

Dear RSC Chemical Biology,

Please find our Revised Review. We have uploaded all the required documents and files. One point, because of the space limitation, we have removed the figures from the manuscript. All the figures, in high resolution, have been uploaded separately.
For the copyrights for Figure 3, 8, 9 and 10 have been uploaded. Dr. Ploegh is the original author of Figure 4 and 7 and thus doesn't require copyright permission. Figure 5 and 6 are open access articles and do not have copyright either for non profit usage.

We hope everything is set, but if anything is missing please let us know.

Best,
Thibault Harmand

Dear Reviewers,
Thank you for taking the time to look over our manuscript. Your feedback is really appreciated.
Reviewer 1
Pg 5: As more relevant references for the deep tissue penetration of nanobodies, the authors could refer to the following papers: - Hernández, 2019, Mol Imaging Biol - Debie, 2020, J Control Release
 The 2 references have been added
Pg 7: "This is often accompanied by uptake of the imaging agent in the kidneys and the liver." Normally, hepatic clearance is not expected with Nanobodies, so I propose to remove the last part of the sentence. Liver uptake is only seen in case of constitutive expression of the target in liver tissue, or in case the imaging label negatively affects the nanobody's biodistribution.
 The last part of the sentence has been removed as suggested
Pg 13: 99mTC --> 99mTc
 Correction made
Pg 15: "Despite a slightly higher accumulation in the bladder" --> Accumulation in the bladder is not relevant since this highly depends on whether the mouse has urinated just before being subjected to an imaging scan or not.
 This part has been removed
Table 3: The IRDye800CW-labeled anti-CEA nanobody misses in this table as image-guided surgery agent. (Lwin, 2020, Surgery)
 The reference has been added to the table
Reviewer 2
- Page, please include a reference for 99mTc-labeling of a His6-tag.
 Two references have been added
- Figure 2 A and C, shown are various widely used chelators and dyes. However, since these are discussed in the context of their covalent attachment to nanobodies, please provide at least some of the structures as representative reagents for the bioconjugation reactions, for example as a maleimide reagent etc.. This information is useful for the reader to see the typical reagents and to get a feeling about which parts of the molecules can be modified for the attachment.
 The chelators and fluorescent dyes have been modified to display some of the most commonly found reactive handles like maleimide, NHS ester, etc. The legend of this figure has also been slightly modified to take your comment into account.
- Figure 3, the differences between the solid, dashed and dotted lines are very hard to appreciate
 The figure has been modified to make the distinction clearer
- Please re-iterate on the renal clearance issue associated with nanobodies. On the one hand, the advantages are highlighted, i.e., yielding a high signal-to-background ratio for imaging already 1 h after nanobody administration. On the other hand, renal uptake can be problematic for several reasons and can be modulated (by sequence, by PEG). However, would alleviating the latter problem mean that one loses again the advantage of the fast clearance? Can sequences and PEG also influence the clearance from the kidney, such that both could be accomplished, fast clearance from the circulation and fast clearance from the kidney?
 This drawback of nanobodies has been re-emphasize in the conclusion of the review
- Page 10/11, include reference to Figure 4
 This has been corrected
- A few typos, e.g., biorthogonal > bioorthogonal, 650nm > 650 nm
Typos have been corrected

02-Mar-2021

Dear Dr Ploegh:

Manuscript ID: CB-REV-02-2021-000023.R1
TITLE: Nanobodies: Powerful Tools For In Vivo, Non-Invasive, Imaging

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

All my points have been addressed by the authors.