A Novel Anti-Cadherin-17 Monoclonal Antibody, Ca₁₇Mab-5, for Multiple Applications

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study reports the development of a new anti-CDH17 monoclonal
antibody, Ca17Mab-5, generated using cell-based immunization and flow cytometry-
based screening. The antibody specifically recognizes CDH17-overexpressing CHO
cells, detects endogenous CDH17 in colorectal cancer cell lines, shows no detectable
cross-reactivity with 21 other cadherins, detects CDH17 by Western blotting, and stains
normal colon epithelium and colorectal cancer tissues by IHC.
The manuscript’s main strength is the demonstration that Ca17Mab-5 works across
multiple platforms: flow cytometry, Western blotting, and FFPE immunohistochemistry.
This versatility is relevant for both basic research and diagnostic pathology. The
comparison with the commercial clone CDH17/2618 is also useful, particularly because
Ca17Mab-5 appears to show superior flow cytometric reactivity and less background
staining in CHO-K1 controls. 
Major comments
1. The therapeutic potential of Ca17Mab-5 is overstated relative to the data
presented.
The abstract and conclusion state that Ca17Mab-5 has potential for tumor diagnosis and
therapy. However, the manuscript does not present functional therapeutic data. There
are no ADCC assays, CDC assays, internalization studies, ADC payload-delivery
experiments, CAR-T/CAR-NK experiments, tumor-growth inhibition assays, or in vivo
efficacy data.
The data support Ca17Mab-5 as a research and diagnostic detection antibody, but not
yet as a therapeutic antibody. The authors do mention future generation of mouse IgG2a
or human IgG1 formats for ADCC and xenograft studies, which is appropriate. The
abstract and conclusion should therefore be revised to state that Ca17Mab-5 may serve
as a platform for future therapeutic development, rather than implying current
therapeutic potential.
2. Epitope mapping is missing and limits mechanistic and translational
interpretation.
The manuscript emphasizes that CDH17 is therapeutically relevant because antibodies
may block CDH17 interactions, support ADC development, or help generate cancer-
specific antibodies. However, the epitope recognized by Ca17Mab-5 is not identified.
The authors themselves state that epitope identification is essential to investigate
neutralizing activity and biological effects. 
At minimum, the authors should discuss this limitation more explicitly. Ideally, epitope
mapping should be added, especially because CDH17 contains seven extracellular
cadherin repeats and functionally important regions such as the RGD motif involved in
α2β1 integrin interaction. Without epitope mapping, it is not possible to determine
whether Ca17Mab-5 is likely to interfere with biologically relevant CDH17 interactions.
3. The IHC analysis should be more quantitative and clinically interpretable.

The IHC data are promising. Ca17Mab-5 stained normal colon epithelium strongly and
detected CDH17 in 133 of 154 colorectal cancer cases. The representative tissue
microarray images on page 11 are clear and show a range of staining intensities from 3+
to negative. 
However, the IHC analysis remains descriptive. The authors should provide a more
rigorous scoring summary across all tissue microarrays, including the number and
percentage of cases scored 0, 1+, 2+, and 3+. If clinicopathologic information is
available, staining should be correlated with differentiation status, TNM stage,
histological subtype, and normal versus tumor tissue. The manuscript notes that staining
“tended to be reduced” in poorly differentiated adenocarcinomas, but this should be
supported by quantitative data or softened. 
4. Specificity testing is strong within the cadherin family but incomplete for
broader diagnostic use.
The authors convincingly show no detectable cross-reactivity with 21 other cadherins
using cadherin-overexpressing CHO-K1 cells. This is a major strength. However, if the
antibody is proposed as a diagnostic tool, broader tissue specificity should be assessed.
CDH17 is expected to be largely restricted to intestinal epithelium, but diagnostic
implementation would benefit from staining a normal-tissue panel and a wider range of
tumor types, especially gastric, pancreatic, hepatocellular, ovarian, and pancreatic
neuroendocrine tumors, all of which are discussed as CDH17-expressing malignancies.
This is particularly important because normal colon epithelium shows strong staining,
raising the need to define the full normal-tissue expression profile and possible
limitations for therapeutic targeting.
Minor comments
1. Several typographical and grammatical errors should be corrected.
Examples include “whearas” instead of “whereas,” “sperior” instead of “superior,” and
“both Ca17Mab-5 showed” instead of “Ca17Mab-5 showed.” The manuscript would
benefit from a careful English-language edit.
2. The antibody-generation strategy should report more detail.
The study states that 129 of 474 wells were positive in screening, 10 anti-CDH17 mAb-
producing clones were established, and Ca17Mab-5 was selected because it worked in
all applications. This is useful, but the authors should briefly summarize how the other
nine clones performed. A supplementary table comparing the 10 clones across flow
cytometry, Western blotting, and IHC would strengthen the rationale for selecting
Ca17Mab-5.
3. Binding affinity interpretation should be refined.
The reported KD values for Ca17Mab-5 were approximately 1.4 × 10−8 M for
CHO/CDH17 and 1.3 × 10−8 M for COLO205, which the authors describe as

“moderate binding affinity.” This is reasonable, but the authors should clarify that these
are apparent affinities measured on intact cells by flow cytometry, not purified antigen
binding affinities. Cell-surface density, secondary antibody signal, and avidity effects
can influence these estimates.
4. Discussion of on-target/off-tumor toxicity is appropriate but should be
connected more directly to Ca17Mab-5.
The authors correctly note that CDH17 is expressed on normal intestinal epithelium and
that adverse effects such as diarrhea and mucositis may limit the therapeutic window of
CDH17-targeted approaches. This is an important point. The manuscript should make
clear that Ca17Mab-5 currently does not demonstrate cancer-selective binding; it stains
normal colon strongly. Therefore, any therapeutic development would require careful
engineering, dosing strategy, cancer-selective epitope discovery, or payload/modality
optimization.
Recommendation
Minor to moderate revision.
The manuscript presents a useful new anti-CDH17 monoclonal antibody with good
specificity among cadherins and applicability to flow cytometry, Western blotting, and
IHC. The core technical data support publication. Before acceptance, the authors should
moderate therapeutic claims, provide a more quantitative IHC summary, clarify the
limitations of affinity measurement and specificity testing, and correct language and
typographical issues. Additional epitope mapping and broader normal/tumor tissue
validation would substantially strengthen the manuscript, although these could be
framed as future work if the journal’s scope permits a technical antibody-
characterization report.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors describe the generation and characterization of a novel monoclonal antibody against human Cadherin-17 (CDH17), designated Ca17Mab-5, using their Cell-Based Immunization and Screening (CBIS) platform. Following immunization with CDH17-overexpressing cells and hybridoma screening by flow cytometry, the authors selected a clone that recognizes cell surface CDH17 with high specificity. The antibody was evaluated by flow cytometry, Western blotting, and immunohistochemistry using engineered cell lines, colorectal cancer cell lines, formalin-fixed paraffin-embedded cell blocks, and colorectal cancer tissue microarrays. The authors conclude that Ca17Mab-5 displays superior specificity and broader applicability than a commercially available anti-CDH17 antibody, making it a useful reagent for both basic research and future diagnostic or therapeutic applications.

Major Comments

Major Comment 1

The central claim that Ca17Mab-5 is superior to the commercially available antibody CDH17/2618 is only partially supported by the presented data (Figures 2, 4, 6, and 7). The comparison relies largely on representative flow cytometry histograms and qualitative immunohistochemical images. Quantitative comparisons of signal intensity, signal-to-background ratio, sensitivity, or specificity are not provided. Without objective metrics, the extent of the claimed improvement remains difficult to assess.

Major Comment 2

The specificity study presented in Figure 3 is one of the strongest parts of the manuscript. However, specificity is evaluated exclusively using cadherin-overexpressing CHO cells. This demonstrates the absence of cross-reactivity within the cadherin family but does not exclude recognition of unrelated proteins expressed in human tissues. The authors should acknowledge this limitation explicitly when discussing antibody specificity.

Major Comment 3

The affinity measurements indicate dissociation constants in the low nanomolar range, but only three independent measurements are reported, and no statistical comparison with the commercial antibody is provided (Figure 5). Since the manuscript argues that Ca17Mab-5 performs better than CDH17/2618, a direct quantitative comparison would substantially strengthen this conclusion. At present, the superiority claim is supported primarily by qualitative observations.

Major Comment 4

The Discussion repeatedly emphasizes the therapeutic potential of Ca17Mab-5 for ADCs, bispecific antibodies, CAR-T cells, and other immunotherapeutic modalities. These statements are speculative because no experiments addressing receptor internalization, epitope accessibility, antibody-mediated effector functions, or in vivo antitumor activity were performed. The current data establish that Ca17Mab-5 is an effective detection antibody, but they do not demonstrate suitability for therapeutic development. The Discussion should distinguish more clearly between demonstrated findings and future possibilities.

Major Comment 5

The manuscript does not identify the epitope recognized by Ca17Mab-5. This limitation becomes particularly relevant because the Discussion proposes future therapeutic applications. Epitope location determines accessibility, internalization, competition with endogenous ligands, and compatibility with therapeutic engineering. Although epitope mapping is not essential for publication, its absence should be acknowledged more explicitly as a limitation of the current study.

Major Comment 6

The immunohistochemical analysis demonstrates positive staining in 86% of colorectal cancer samples, but the presentation remains descriptive (Figure 8 and Table 1). The manuscript would benefit from quantitative analysis examining the relationship between staining intensity and clinicopathological variables, including tumor differentiation or TNM stage. Even if statistical associations are not significant, reporting the analysis would strengthen the biological interpretation.

Major Comment 7

Statistical reporting throughout the manuscript is limited. Apart from the affinity measurements, the study contains very little quantitative analysis. The manuscript should clearly define biological replicates, specify how many independent experiments were performed for each assay, report the statistical methods where applicable, and indicate whether the presented data are representative or pooled from independent experiments.

Minor Comments

Minor Comment 1

The Introduction would benefit from a clearer distinction between the physiological role of CDH17 in intestinal homeostasis and its pathological role in colorectal cancer progression. This would improve the logical flow leading to the rationale for antibody development.

Minor Comment 2

Several statements in the Discussion use language implying therapeutic efficacy, whereas the experimental data demonstrate analytical performance only. More conservative wording would improve scientific precision.

Minor Comment 3

Figure legends should explicitly define the number of biological replicates performed for each experiment. This information is currently incomplete.

Minor Comment 4

The manuscript would benefit from reporting confidence intervals for the estimated KD values in addition to the reported mean ± standard deviation.

Minor Comment 5

The tissue microarray analysis could be strengthened by providing a concise summary table reporting staining intensity across tumor grades in addition to the individual case listing.

Minor Comment 6

Several typographical errors should be corrected. Examples include “whearas” in the Results section and “sperior reactivity,” which should read “superior reactivity.”

Minor Comment 7

The authors cite numerous ongoing clinical trials targeting CDH17. Including a brief table summarizing the therapeutic modality, target population, and current clinical phase would improve readability.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Accept in present form.

 

Author Response

Thank you very much.

Reviewer 2 Report

Comments and Suggestions for Authors

Major Comments

Major Comment 1 – Partially Resolved

The additional experiments using CDH17-knockout cells strengthen the specificity argument. However, the manuscript still concludes that Ca17Mab-5 is superior to the commercial antibody primarily on the basis of qualitative observations. The inability to determine the KD of CDH17/2618 does not demonstrate superior affinity or overall analytical performance. The authors should moderate statements claiming superiority and instead conclude that Ca17Mab-5 exhibits excellent specificity and broad applicability under the experimental conditions tested.

Major Comment 3 – Partially Resolved

The explanation regarding the inability to calculate the KD of CDH17/2618 is scientifically reasonable. Nevertheless, the manuscript should explicitly acknowledge that a direct quantitative affinity comparison between the two antibodies could not be performed. Consequently, claims suggesting superior performance should not be based on affinity considerations.

Minor Comments

Minor Comment 4 – Not Fully Resolved

Rather than asking how confidence intervals should be reported, the authors should simply include the 95% confidence intervals for the fitted KD values in the manuscript or Supplementary Information whenever available. This would improve the rigor and transparency of the affinity analysis.

Minor Comment 1

The manuscript would benefit from slightly more cautious wording when discussing the analytical advantages of Ca17Mab-5. Throughout the Discussion, statements referring to its improved performance should consistently reflect the scope of the experimental evidence presented.

Minor Comment 2

The authors state that no association was observed between IHC score and T stage. Although this negative result is informative, it would improve transparency to briefly report the statistical test performed and include the corresponding result (e.g., P value) either in the Results or as supplementary information.

Minor Comment 3

The authors indicate that information on tumor differentiation and grade was unavailable for the tissue microarrays. This limitation should be explicitly acknowledged in the Discussion, as it restricts the biological interpretation of the immunohistochemical findings.

Minor Comment 4

The figure legends now specify the number of independent experiments, which improves reproducibility. However, the Methods section would also benefit from a brief statement clarifying whether the reported experiments represent independent biological replicates or technical replicates.

The revised manuscript is substantially improved, and most of the concerns raised during the initial review have been satisfactorily addressed. The remaining issues are primarily related to interpretation and presentation rather than experimental design. Addressing the points above would further improve the scientific rigor and ensure that the conclusions remain fully supported by the available data.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf