From the journal RSC Chemical Biology Peer review history

05-May-2023

Dear Dr Nishimura:

Manuscript ID: CB-ART-03-2023-000036
TITLE: Structural and molecular insight into antibody recognition of dynamic neoepitope in membrane tethered MUC1 of pancreatic cancer cells and secreted exosomes

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Professor Zaneta Nikolovska-Coleska
Associate Editor, RSC Chemical Biology

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Reviewer 1

Prof. Nishimura and coworkers have performed a structural and molecular insight into the anti-MUC1 antibody (SN-131). This antibody binds specifically to core 1 but not core 2 type O-glycans found in normal cells. SN-131 shows the highest affinity towards MUC1-bearing ST antigen at immunodominant DTR motif (K D = 1.58 nM) independent of the glycosylation states of other Ser/Thr residues in the MUC1 tandem repeats. X-ray structure revealed that SN-131 interacts directly with Neu5Ac and root GalNAc of the ST antigen and the proximal peptide region. Pancreatic cancer cells and secreted exosomes express tumor associated MUC1, which this antibody recognizes. Therefore, the results shown in this paper may be a promising therapeutic strategy for improving the treatment outcome of patients with pancreatic cancer.
In general, the paper is clear and well-written. However, some significant concerns must be addressed before publication:

1) In the X-ray structure, the electron density around the Neu5Ac residue, particularly around O9, is relatively poor (Figure 3b). It would be interesting to perform molecular dynamics (MD) simulations to determine the stability of the hydrogen bonding between O9 of the Neu5Ac moiety and Asn35 of the light chain of the antibody. In this line, the affinity of compounds 3 and 4 is very similar. What is the KD for a glycopeptide with antigen T? In this regard, showing the SPR data of glycopeptides 7 and 8 (or similar derivatives without the 5-oxo function) would be helpful.

2) According to Figure 5d, antibody SN-131 recognizes a "turn-like" conformation of the peptide backbone, which is not the most populated one in solution (see reference 20). However, the thermodynamic parameters determined for MUC1-Tn shown in Fig. S4 does not indicate this entropy penalty, which is evident in the naked peptide. Therefore, the authors should explain in more detail these results. In the same line, what is the conformation of the glycosidic linkage in the bound state? Is the same conformation as in the free form of the glycopeptide in water?

3) Don’t the authors have any problem with the Cys residue of compounds 7-9 in the microarray studies? The protocols don’t use TCEP of similar reagents to avoid disulfide bridge formation.

4) It would be interesting to show a negative control for the flow cytometry analysis and live cell images in Figures 4a and 4b, respectively.

Minor points:

1) The structure of the article, mixing the figure captions with the main text, makes it difficult to follow it.
2) Enhance the quality of Figures 2 and 4. These figures contain too much information, which isn't easy to follow. Figure 2c and Figure 4g could be moved to the SI. Figure S1b should replace Figure 2d.

Reviewer 2

The authors present an outstanding example of the application of a multidisciplinary approach to unravel the recognition features of an anti-MIC 1 antibody in different environments. The research is at the frontiers between chemistry and biology and involves different cell biology, chemical biology, and structural biology techniques at the maximum level. The work with exosomes is highly relevant and opens new avenues in the field.

May 10, 2023
Dear: Professor Zaneta Nikolovska-Coleska
Associate Editor, RSC Chemical Biology
We would like to submit a revised manuscript (CB-ART-03-2023-000036) entitled as, “Structural and molecular insight into antibody recognition of dynamic neoepitope in membrane tethered MUC1 of pancreatic cancer cells and secreted exosomes” to RSC Chemical Biology as an Article.
According to valuable comments and suggestions from your reviewers and you, we carefully revised our manuscript. The followings are changes made:
To Referee: 1
1) “Epitope mapping and SPR analysis demonstrated that SN-131 exhibits the highest affinity (KD = 1.58 nM) with MUC1 glycopeptide bearing ST antigen at Asp-Thr-Arg-Pro moiety (4), an essential glycopeptidic epitope (Fig. 2d-2f, Fig. S2).” was changed to “Epitope mapping and SPR analysis demonstrated that SN-131 exhibits high affinities (KD = 3.78 nM and 1.58 nM) with MUC1 glycopeptide bearing Tn and ST antigens at Asp-Thr-Arg-Pro moiety (3) and (4) (Fig. 2d and 2e, Fig. S2). We have confirmed the negligible effect of N-terminal 5-oxo-function on the interaction of antibodies recognizing the immunodominant DTR region when the epitope locates the center with an enough distance from N-terminus.
2) A new sentence, “The GalNAc addition seems to give advantages to the binding by overcoming the entropy-enthalpy compensation problem (Fig. S4). However, we could not find significant hydrophobic interactions between GalNAc and SN-131, suggesting that there is some new factor for decreasing -TS value.” was added and the sentence, “On the other hand, it is not surprising that anti-MUC1/KL-6 mAb and many other anti-MUC1 mAbs reacting with core 2 type O-glycans would be captured predominantly by MUC1 TRD expressed in the normal cells.” was deleted.
3) We did not observe any problem in the microarray studies with the C-terminal Cys residue of glycopeptides tested. However, it seems likely that the use of TCEP would be used to avoid disulfide bridge formation in case for microarray displaying high concentration of glycopeptide derivatives.
4) In Figure 4, a control experiment for live cell imaging was added.
5) The structure of the article (the places of figure captions) was improved.
6) The quality of the figures were improved. Figure 2c was moved to the SI. Figure 2d (newly Figure 2c) was replaced by Figure S1b.
We would like to express our great appreciation to the helpful and important suggestions and comments from your referees and you to improve our manuscript.
Sincerely yours,

Shin-Ichiro Nishimura, PhD
Professor, Graduate School of Life Science and Faculty of Advanced Life Science
Hokkaido University
N21 W11, Sapporo
001-0021, Japan
TEL: +81-11-706-9043, FAX: +81-11-706-9042
Email: shin@sci.hokudai.ac.jp

19-May-2023

Dear Dr Nishimura:

Manuscript ID: CB-ART-03-2023-000036.R1
TITLE: Structural and molecular insight into antibody recognition of dynamic neoepitope in membrane tethered MUC1 of pancreatic cancer cells and secreted exosomes

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Professor Zaneta Nikolovska-Coleska
Associate Editor, RSC Chemical Biology

Reviewer 1

The authors have substantially improved the manuscript and effectively addressed all of my concerns.