Establishment of a Novel Anti-CD44 Variant 10 Monoclonal Antibody C44Mab-18 for Immunohistochemical Analysis against Oral Squamous Cell Carcinomas
Round 1
Reviewer 1 Report
The manuscript entitled "Establishment of A Novel Anti-CD44 Variant 10 Monoclonal 2 Antibody C44Mab-18 for Immunohistochemical Analysis 3 against Oral Squamous cell carcinomas" contributed by Ishikawa et al. provided a report about mice monoclonal antibodies against CD44 C44Mab-18 in CD44 detection and also in oral cancer tissue microarray IHC.
In the introduction, the authors use HNSCC than OSCC in the first section. Please correct it.
Please explain the difference and significance of this manuscript which has a vary similar article structure and experiment approaches in reference 28 "antibodies-12 -00031"
Please discuss why the CD44-v3-10 always has the similar blotting pattern in both papers.
Good enough
Author Response
The manuscript entitled "Establishment of A Novel Anti-CD44 Variant 10 Monoclonal 2 Antibody C44Mab-18 for Immunohistochemical Analysis 3 against Oral Squamous cell carcinomas" contributed by Ishikawa et al. provided a report about mice monoclonal antibodies against CD44 C44Mab-18 in CD44 detection and also in oral cancer tissue microarray IHC.
In the introduction, the authors use HNSCC than OSCC in the first section. Please correct it.
OSCC is one type of HNSCC. In the introduction, we mainly cited the information on HNSCC which includes not only OSCC, but also other SCC derived from the nasal cavity, oropharynx, hypopharynx, etc.
Please explain the difference and significance of this manuscript which has a vary similar article structure and experiment approaches in reference 28 "antibodies-12 -00031"
We reported anti-CD44 mAbs that recognize different variants. Each variant possesses a unique function and expression pattern in tumors. Therefore, we reported in several papers.
Furthermore, we explained the significance in the conclusion session, as follows.
5. Conclusion
In this study, we established an anti-CD44v10 mAb (C44Mab-18). We also established an anti-CD44v8 mAb (C44Mab-94) (manuscript submitted, see Supplementary Materials). Therefore, we have established the anti-CD44 mAb library that covers almost CD44 variants. The library could contribute to the diagnosis of not only carcinoma, but also hematopoietic malignancies. Since we have already cloned the VH and VL cDNA of anti-CD44 mAbs, the production of recombinant mAbs or CARs could contribute to the development of novel tumor therapies.
Please discuss why the CD44-v3-10 always has the similar blotting pattern in both papers.
Because CHO/CD44-v3-10 was transfected with the cDNA of CD44v3-10, the transcribed mRNA never received the alternative splicing. Therefore, CHO/CD44-v3-10 produced only CD44-v3-10, but not other splicing variants.
Reviewer 2 Report
In this study, Ishikawa and colleagues developed a monoclonal anti-CD44 antibody specifically detecting variants 3-10 and they evaluated its applications for western blot, flow cytometry and immunohistochemistry assays in OSCC samples. It is well established that CD44 has several variants generated by alternative splicing and this protein is highly accumulated in cancers and it is associated with aggressiveness, resistance to therapy and cancer stem-like cells. Therefore, developing antibodies that recognize different isoforms of this protein can be useful for clinical applications.
Overall, the experiments were well performed and enough to sustain their hypothesis.
Author Response
In this study, Ishikawa and colleagues developed a monoclonal anti-CD44 antibody specifically detecting variants 3-10 and they evaluated its applications for western blot, flow cytometry and immunohistochemistry assays in OSCC samples. It is well established that CD44 has several variants generated by alternative splicing and this protein is highly accumulated in cancers and it is associated with aggressiveness, resistance to therapy and cancer stem-like cells. Therefore, developing antibodies that recognize different isoforms of this protein can be useful for clinical applications.
Overall, the experiments were well performed and enough to sustain their hypothesis.
Thank you very much for the comment.
Reviewer 3 Report
The study conducted by the authors successfully demonstrates the specific recognition of CD44 v10 by the newly developed monoclonal antibody, C44Mab-18. While the manuscript presents compelling data that highlight the specificity and usefulness of this antibody, there are a few areas that could benefit from improvement in order to enhance overall comprehension.
Introduction:
The rationale behind the creation of the CD44 v10 antibody remains somewhat unclear. Although the authors have previously generated monoclonal antibodies against other CD44 variants, it would be valuable for them to provide further details regarding the specific objectives of developing the CD44 v10 antibody. This clarification would help readers understand whether this antibody was created as part of a larger project aimed at building a comprehensive library of CD44 variants or if it serves a distinct purpose. Additionally, providing additional information on the expression and function of each CD44 variant would contribute to a more comprehensive understanding.
Results and Discussion:
3.1: In order to avoid confusion between the authors' past and current studies, it is recommended that they clearly distinguish between the two. For example, phrases such as "In our previous work" can be employed to refer to earlier studies. Alternatively, the authors may choose to exclude any references to past studies in the results section altogether.
3.3: The authors mention that Mab-18 exhibited moderate binding affinity. It would be informative to compare this binding affinity to that of other monoclonal antibodies targeting CD44 (including pan and other variants) to provide a context for evaluating its affinity.
Table 1: The staining intensity is categorized as -, +, ++, or +++. It would be beneficial for the authors to explain the criteria used for these ratings or if there was a specific quantification system employed. Moreover, clarification is needed regarding whether the entire tumor tissue section or only specific areas were assessed. Additionally, as Mab-46 consistently demonstrated higher signal intensity, it would be valuable to explain if this is attributed to its ability to stain stromal tissue.
The discussion section would benefit from a dedicated conclusion section, wherein the authors summarize the significance and utility of the newly developed CD44 v10 monoclonal antibody. Additionally, it would be valuable to elaborate on potential future applications of this antibody to enhance its practical implications.
Author Response
The study conducted by the authors successfully demonstrates the specific recognition of CD44 v10 by the newly developed monoclonal antibody, C44Mab-18. While the manuscript presents compelling data that highlight the specificity and usefulness of this antibody, there are a few areas that could benefit from improvement in order to enhance overall comprehension.
Introduction:
The rationale behind the creation of the CD44 v10 antibody remains somewhat unclear. Although the authors have previously generated monoclonal antibodies against other CD44 variants, it would be valuable for them to provide further details regarding the specific objectives of developing the CD44 v10 antibody. This clarification would help readers understand whether this antibody was created as part of a larger project aimed at building a comprehensive library of CD44 variants or if it serves a distinct purpose. Additionally, providing additional information on the expression and function of each CD44 variant would contribute to a more comprehensive understanding.
According to the comments, we added the following statement in introduction.
“The generation of anti-CD44 mAbs which recognize all variant exons is important for the comprehensive analysis for human tumors.”
Results and Discussion:
3.1: In order to avoid confusion between the authors' past and current studies, it is recommended that they clearly distinguish between the two. For example, phrases such as "In our previous work" can be employed to refer to earlier studies. Alternatively, the authors may choose to exclude any references to past studies in the results section altogether.
We added “In our previous work” in introduction, results, and discussion parts.
3.3: The authors mention that Mab-18 exhibited moderate binding affinity. It would be informative to compare this binding affinity to that of other monoclonal antibodies targeting CD44 (including pan and other variants) to provide a context for evaluating its affinity.
C44Mab-18 is IgM antibody. In contrast, our established anti-CD44 mAbs are IgG1. In our binding affinity analysis, it is difficult to compare the affinity between IgG and IgM. Therefore, we cloned the VH and VL cDNA of C44Mab-18 and produced the class switched (IgG1) recombinant Ab. We are going to measure the KD and compare to other anti-CD44 mAbs.
Table 1: The staining intensity is categorized as -, +, ++, or +++. It would be beneficial for the authors to explain the criteria used for these ratings or if there was a specific quantification system employed. Moreover, clarification is needed regarding whether the entire tumor tissue section or only specific areas were assessed. Additionally, as Mab-46 consistently demonstrated higher signal intensity, it would be valuable to explain if this is attributed to its ability to stain stromal tissue.
We do not use a specific quantification system. We focus on the expression in tumor, but not stroma cells.
Under the microscope, we determine the intensity of staining, as follows.
-, No stain; +, Weak intensity; ++, Moderate intensity; +++, Strong intensity.
The discussion section would benefit from a dedicated conclusion section, wherein the authors summarize the significance and utility of the newly developed CD44 v10 monoclonal antibody. Additionally, it would be valuable to elaborate on potential future applications of this antibody to enhance its practical implications.
We added the conclusion section to summarize our research, as follows.
5. Conclusion
In this study, we established an anti-CD44v10 mAb (C44Mab-18). We also established an anti-CD44v8 mAb (C44Mab-94) (manuscript submitted, see Supplementary Materials). Therefore, we have established the anti-CD44 mAb library that covers almost CD44 variants. The library could contribute the diagnosis of not only carcinoma, but also hematopoietic malignancies. Since we have already cloned the VH and VL cDNA of anti-CD44 mAbs, the production of recombinant mAbs or CARs could contribute the development for novel tumor therapies.
