Ca₁₃Mab-17, a Novel Anti-Cadherin-13 Monoclonal Antibody for Versatile Applications

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is technically sound and follows the standard format for an MDPI antibody characterization paper. Overall, I think this manuscript is suited for Antibodies after addressing the following points to clarify the methodology and results.

Major:

1. In Figure 6, it is interesting to note that the antibody binds to both the mature and precursor forms. Based on the WB result, we can conclude that the epitope is located within the mature EC domain. However, in the WB result, the two bands showed different intensities. This result can lead to two possibilities: either the precursor form is lower than the mature form in the cell lysate, or the prodomain might hinder epitope exposure. Given that the Ca13Mab-17 binds both the mature and uncleaved forms, the exact location of the epitope is of great interest. I would suggest that the authors use a structural prediction method (e.g., AlphaFold Multimer or docking tools) to identify the potential binding region. This would significantly strengthen the paper by providing a structural rationale for the antibody's versatile performance across various CDH13 forms. Note that this is not strictly required for this characterization study, but such a model could provide a solid structural hypothesis.
2. The comparison with the commercial clone 392411 is a highlight of the paper. Based on Figure 5, it is clear that the commercial clone 392411 showed stronger binding affinity than Ca13Mab-17, with a low nanomolar affinity. However, in section 3.2, the authors state that 'Additionally, a commercially available anti-CDH13 mAb (clone 392411) did not recognize CHO/CDH13.' Could the authors speculate why this occurs? This would help readers understand the advantages of Ca13Mab-17 compared with 392411.
3. The authors state that 'The CBIS method involves high-throughput flow cytometry–based screening, and mAbs produced by this method typically recognize conformational epitopes, enabling their use in flow cytometry.' However, I suggest the authors add a Western blot experiment using Ca13Mab-17 against CDH13 under both reducing and non-reducing conditions to determine whether the antibody recognizes a linear epitope or a strictly conformational one.
4. The authors noted that CDH13, a tumor suppressor, is upregulated in cancers, including ccRCC. Given that one of the major benefits of this antibody is its ability to bind (or potentially distinguish) between the mature and precursor forms, the authors should discuss how this may be relevant for future diagnostic studies.

Minor:

1. For Figure 5, there are no error bars. Without the error bars, we don't know the distribution of the data or its standard deviation.
2. For Table 1, the authors describe the staining intensity using a semi-quantitative scale. While this is a standard approach, it remains subjective. The authors should provide a more detailed description of what constitutes each grade (e.g., 1+ requires a certain percentage of cell positivity, or it is purely based on optical density). Also, the supplemental file should include a figure showing a side-by-side comparison of the 1+, 2+, and 3+ staining stages.

Overall, this is a high-quality technical report for the antibodies journal. I would recommend addressing these issues before proceeding to publication.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled “Ca13Mab-17, a novel anti-Cadherin-13 monoclonal antibody for versatile applications” described the development of an anti-cadherin-13 monoclonal antibody, Ca13Mab-17, for multiple purposes by using the Cell-Based Immunization and Screening (CBIS) method. Ca13Mab-17 can pick up ectopically expressed and endogenous cadherin-13 in cancer cell lines without cross-reactivity with other cadherins, recognize both the precursor and mature forms of CDH13 in western blotting, and identified CDH13 in new blood vessels and glioblastoma cells by immunohistochemistry. The following are my comments and suggestions:

1. In Figure 3, specificity assay of Ca13Mab-17 for CDH14 was missing.
2. In Figure 7, it appears that Ca13Mab-17 has strong cytoplasmic staining in addition to the membranous staining. Please explain why Ca13Mab-17 can have cytoplasmic staining in addition to membranous staining for CDH13.
3. The commercial anti-CDH13 antibody (MAB3264, clone 392411) can work in flow cytometry, immunohistochemistry, and Western blotting analyses. What is the difference between Ca13Mab-17 and 392411?

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The work by Shimizu et al. reports the development of a novel anti-human CDH13 monoclonal antibody generated by immunizing mice with the mature form of CDH13-overexpressing LN229 cells. Among the obtained clones, Ca13Mab-17 specifically recognizes CDH13-overexpressing Chinese hamster ovary (CHO-K1) cells. This result is particularly interesting, as the antibody shows no detectable cross-reactivity with 21 other cadherins and is able to recognize both the mature and uncleaved precursor forms of CDH13 in human glioblastoma and lung mesothelioma cell lines. Given the potential role of CDH13 as a tumor biomarker, this study presents a promising tool for tumor diagnosis and thereby I recommend publication after minor revisions.

Specific comments:

Introduction (line 69):

The authors state that monoclonal antibodies capable of detecting CDH13 by Western blotting (WB) or immunohistochemistry (IHC) have been developed, whereas suitable antibodies for flow cytometry are limited.

Please clarify why the development of antibodies suitable for flow cytometry is particularly important in the context of prognostic markers.

What advantages does flow cytometry offer compared to WB or IHC in this setting?

Results (line 209 / Figure 2):

The authors report that Ca13Mab-17 detects CDH13 on CHO cells in a dose-dependent manner, whereas the commercially available antibody (clone 392411) does not. However, these comparative data are not shown, and no reference is provided.

Please include the supporting data or cite an appropriate reference.

Figure 4 and related interpretation:

The authors compare Ca13Mab-17 with clone 392411 and show that both antibodies can detect CDH13 on three different cell lines by flow cytometry. However, the introduction suggests that no antibodies are available for CDH13 detection by flow cytometry.

This appears inconsistent with the presented results. It would be more accurate to state that limitations may exist depending on the cell system (e.g., CHO cells).

Why is Ca13Mab-17 uniquely effective in CHO/CDH13 cells? What distinguishes CDH13 expression or presentation in CHO cells compared to other cell types?

Considering that the intended application is tumor diagnosis, what is the advantage of Ca13Mab-17 over clone 392411 in clinically relevant tumor cells, especially if the commercial antibody shows higher affinity?

Figure 6 (Western blot):

Please include appropriate loading controls.

Additionally, why does RcMab-1 produce a band in CHO-K1 cells?

Discussion (line 316):

The authors suggest that Ca13Mab-17 could be useful for isolating CDH13-positive cells via fluorescence-activated cell sorting (FACS). This is a key strength of the study, particularly given the antibody’s specificity.

However, to support this claim, the authors should provide evidence that clone 392411 is less specific than Ca13Mab-17.

Discussion (line 326):

The authors state that clone 392411 could not detect the uncleaved precursor form of CDH13 in LN229 and U87MG lysates by WB.

These results are not shown. Please include the corresponding data or clarify where they are presented.  

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have fully addressed my comments.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors addressed all the points raised by the reviewer