Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors did a great job comparing the THP vs. macrocyclic NOTA for Ga-labeling of a B7-H3 affibody. They showed that THP enabled rapid labeling at RT with near-quantitative yield, while NOTA requires heating and gives lower yield. In vitro specificity and relative affinity appear comparable, and in vivo biodistribution in SKOV3 showed similar tumor uptake. Overall, the study addresses practical radiochemistry considerations for translating B7-H3 affibody PET tracers. 

Major comments: 

1. Clarifying novelty and translational focus
To strengthen the framing, the authors could more clearly state what is new in this manuscript and why the use of THP is expected to aid clinical translation beyond improved radiochemical yield. A brief paragraph near the end of the Introduction defining the specific gap addressed, and a more explicit discussion of practical advantages (e.g., simplified workflow, reduced preparation time, no heating or purification) would improve clarity.

2. Head-to-head biodistribution design and statistics
The manuscript describes a “head-to-head” biodistribution comparison, but the experimental design is not fully clear. It would be helpful to clarify whether the same animals received both tracers (paired vs. crossover design) or whether separate groups were used. This information is important for evaluating the appropriateness of the statistical analysis.

3. Interpretation of normalized binding data
In vitro binding data are presented in normalized form. While this facilitates comparison of trends, it is unclear whether the absolute binding levels corresponding to the normalized values were similar between the THP- and NOTA-conjugated tracers. Clarifying this point, either by providing absolute values or stating their comparability, would strengthen the interpretation.

Minor comment:

there was one B7-B3 typo in the article, and also add n=x in all the figure annotations. 

 

Author Response

Dear Reviewer,

We would like to sincerely thank you for the time and effort you devoted to reviewing our manuscript. We greatly appreciate your careful evaluation and constructive comments, which have been very helpful in improving the quality, clarity, and scientific rigor of our work.

Your insights and suggestions were carefully considered, and we believe they have significantly strengthened the manuscript. Below, we provide a point-by-point response to each of your comments.

Thank you again for your valuable input and contribution to the review process.

Kind regards,
Maryam Oroujeni, PhD

Reviewer 1:

The authors did a great job comparing the THP vs. macrocyclic NOTA for Ga-labeling of a B7-H3 affibody. They showed that THP enabled rapid labeling at RT with near-quantitative yield, while NOTA requires heating and gives lower yield. In vitro specificity and relative affinity appear comparable, and in vivo biodistribution in SKOV3 showed similar tumor uptake. Overall, the study addresses practical radiochemistry considerations for translating B7-H3 affibody PET tracers.

Answer. Thank you for your positive feedback on our study.

Major comments:

1. Clarifying novelty and translational focus

To strengthen the framing, the authors could more clearly state what is new in this manuscript and why the use of THP is expected to aid clinical translation beyond improved radiochemical yield. A brief paragraph near the end of the Introduction defining the specific gap addressed, and a more explicit discussion of practical advantages (e.g., simplified workflow, reduced preparation time, no heating or purification) would improve clarity.

Answer. Thank you for the important point, which will improve our paper! To address this comment, we added the following text to the Introduction section: ‘’Although ⁶⁸Ga-based radiopharmaceuticals are increasingly used, the majority of commonly used chelators still require heating, long labeling times, and post‑labeling purification, which complicates routine clinical implementation. The use of THP chelator offers a distinct advantage by enabling rapid, complexation of ⁶⁸Ga at neutral pH and room‑temperature with high radiochemical yields. This efficient labeling directly addresses a key translational gap: the need for radiotracers that can be prepared reliably and reproducibly in routine clinical settings, including sites without advanced radiochemistry infrastructure. In this manuscript, we build on these advantages of THP to evaluate if THP‑conjugated Affibody molecules can facilitate diagnostic procedures, thereby providing a practical route toward more accessible and scalable clinical translation.”

2. Head-to-head biodistribution design and statistics

The manuscript describes a “head-to-head” biodistribution comparison, but the experimental design is not fully clear. It would be helpful to clarify whether the same animals received both tracers (paired vs. crossover design) or whether separate groups were used. This information is important for evaluating the appropriateness of the statistical analysis.

Answer. Thank you for raising an interesting point for consideration!  To provide more clarification, we have revised the text in 4.6. In Vivo Studies: ‘’Biodistribution of ⁶⁸Ga-labeled Affibody conjugates was measured in BALB/C nu/nu mice bearing B7-H3-positive SKOV-3 xenografts. To establish xenografts, SKOV-3 cells (10⁷ cells/mouse) were subcutaneously injected on the right hind leg of female BALB/c nu/nu mice in two groups of mice (4 mice per each group). For in vivo specificity test, B7-H3-negative Ramos cells (6 × 10⁶ cells/mouse) were subcutaneously implanted on the left hind leg of female BALB/c nu/nu mice (one group of 4 mice). Four mice per group were used in the biodistribution. The biodistribution was measured three weeks after cell implantation. The average animal weight was 18.4±1.3 g. The average tumor weight was 0.2 ± 0.1 g and 0.3 ± 0.1 g for SKOV-3 and Ramos xenografts, respectively. The biodistribution was measured 2 h after injection. Two groups of mice (4 mice for each radiocojugate) were injected with [⁶⁸Ga]Ga-THP-ZB7-H3_2 and [68Ga]Ga-NOTA-ZB7-H3_2 (0.28 nmol, 400 kBq, in 100 µL of PBS) separately, into the tail vein. To test B7-H3-mediated accumulation in vivo, one group of animals (4 mice) bearing B7-H3-negative Ramos xenografts was injected with the same peptide mass and activity of [⁶⁸Ga]Ga-THP-ZB7-H3_2, and the biodistribution was measured 2 h after injection. ‘’

3. Interpretation of normalized binding data

In vitro binding data are presented in normalized form. While this facilitates comparison of trends, it is unclear whether the absolute binding levels corresponding to the normalized values were similar between the THP- and NOTA-conjugated tracers. Clarifying this point, either by providing absolute values or stating their comparability, would strengthen the interpretation.

Answer. Regarding binding specificity, we didn’t normalize values. We have reported the CPM/cell number to have a fairer quantitative comparison between two cell lines and also between two constructs.

Regarding cellular processing, to address the comment, we replaced ‘’Figure 6 ‘’ with non-normalized cellular processing data. Regarding NOTA-conjugated tracer, cellular processing was measured and reported in ref. 24. We added the following text about values for both THP and NOTA-conjugated tracers to the results section as well.”

‘’Total cell-associated bound activity for [⁶⁸Ga]Ga-THP-ZB7-H3_2 was 4.39 ± 0.18 and 6.30 ± 0.38 on SKOV-3 and BT-474, respectively. The internalization rate of [⁶⁸Ga]Ga-THP-Z_{B7-H3_2} was slow, with the internalized fraction accounting for 0.74±0.02 % and 0.70±0.03% in SKOV-3 and BT-474, respectively, after 3 h of incubation.  Total cell-associated bound activity for [⁶⁸Ga]Ga-NOTA-ZB7-H3_2 was 3.80 ± 0.11 and 4.6 ± 0.14 on SKOV-3 and BT-474, respectively. The internalized fraction was similar on both cell lines (0.77±0.11 and 0.75±0.11% of the cell-associated activity in SKOV-3 and BT-474, respectively) [24].’’

Minor comment:

There was one B7-B3 typo in the article, and also add n=x in all the figure annotations.

Answer. Thank you for pointing this out!  Typo was corrected. We agree that it is important to state the numbers. We have reviewed the manuscript and adjusted it when appropriate. For experiments including multiple runs, this has been indicated in the text.

 

Reviewer 2 Report

Comments and Suggestions for Authors

1. The study provides the details of optimizing the labelling chemistry, especially THP V NOTA, rather than introducing any new concept or target. The images are modest with tumor uptake is similar
2. The data show the systemic uptake of THP over NOTA in various organs, but there is no mechanistic explanation through experiments that have been explored. Additional in-vitro assays will strengthen the manuscript.
3. The experiment with PET shows only one time point, which is insufficient to understand kinetics, tumor retention, etc. A time-activity graph is needed to determine if the slight uptake of THP resolves or worsens over time.
4. In the current version, only B7-H3-High and B7-H3-negative are used, which does not reflect the heterogeneity of its expression and TME. The author should include additional positive models to claim the heterogeneity.
5. The author should mention the current ex vivo experimentation related to the B7-H3 receptor densities, along with old published data. Only providing the old data will not help to address how robustly THP reflects actual target expression.
6. Provide full kinetics of binding strength along with IC50 and EC50, which limits the ability to determine if THP alters the interaction or not.
7. The paper mentioned Affibody as a reference and talks more about its safety, but the author fails to discuss whether the THP chelator and labeling condition initiate any risk either in animal study or experimental validations. Concluding the sentence below for THP conjugate affibody “These findings indicate that the THP-conjugated Affibody molecule is a promising candidate for future clinical translation.” would be strong, without providing any dosimetry recordings or comparison with B7-H3imaging.

Author Response

Dear Reviewer,

We would like to sincerely thank you for the time and effort you devoted to reviewing our manuscript. We greatly appreciate your careful evaluation and constructive comments, which have been very helpful in improving the quality, clarity, and scientific rigor of our work.

Your insights and suggestions were carefully considered, and we believe they have significantly strengthened the manuscript. Below, we provide a point-by-point response to each of your comments.

Thank you again for your valuable input and contribution to the review process.

Kind regards,
Maryam Oroujeni, PhD

1-The study provides the details of optimizing the labelling chemistry, especially THP V NOTA, rather than introducing any new concept or target. The images are modest with tumor uptake is similar.

Answer. Yes, this was the goal, as we stated in the Introduction: “This study compared the effects of substituting the macrocyclic chelator NOTA with the acyclic chelator THP on the biodistribution of ⁶⁸Ga-labeled Affibody molecule for imaging B7-H3 expression. Introducing new concepts or targets is always exciting. However, it is important for clinical translation to have an optimal chemistry, in terms of robustness, efficacy, and preserved targeting specificity. It has to be noted that the most successful imaging probes, somatostatin analogues, and PSMA binders underwent a long process of labelling chemistry optimization to make their impact in cancer care. The main point was to improve chemistry, making it simpler, without deteriorating imaging properties.

Our tracer is still much better than alternatives such as sdAb with a tumour uptake of 1.5% ID/g, and tumour-to-blood ratio below 5, tumour-to-muscle ratio around 15 (Funeh CN, Meeus F, Van Winnendael N, De Groof TWM, D'Huyvetter M, Devoogdt N. Design and Preclinical Validation of an Anti-B7-H3-Specific Radiotracer: A Non-Invasive Imaging Tool to Guide B7-H3-Targeted Therapies. Pharmaceuticals (Basel). 2025 Sep 30;18(10):1477); ⁸⁹Zr-antibodies with similar tumour uptake and tumour-to blood ratio but after 96 h after injection, i.e., much later (Fernandes B, Olkowski CP, Ghaemi B, Basuli F, Shi J, Kiesewetter DO, Lang L, Elijah E, Swenson R, Choyke PL, Lin FI, Jacobson O. Unraveling the dynamics of B7-H3-targeting therapeutic antibodies in cancer through PET imaging and antibody pharmacokinetics. J Control Release. 2025 Mar 10;379:478-488. ); 89Zr-DS-antibody (tumour uptake 32.32 ± 6.55 %ID/g on day 7 post injection), much much later (Burvenich, I.J.G.; Parakh, S.; Lee, F.-T.; Guo, N.; Liu, Z.; Gan, H.K.; Rigopoulos, A.; O’Keefe, G.J.; Gong, S.J.; Goh, Y.W.; et al.Molecular imaging of T cell co-regulator factor B7-H3 with 89Zr-DS-5573a. Theranostics 2018, 8, 4199–4209.);  a recently reported data of using a non-affinity matured affibody with lower tumour uptake (below 10% ID%g) but good clinical visualization (Xia L, Wu Y, Ren Y, Wang Z, Zhou N, Zhou W, Zhou L, Jia L, He C, Meng X, Zhu H, Yang Z. A whole-body imaging technique for tumor-specific diagnostics and screening of B7H3-targeted therapies. J Clin Invest. 2025 Jan 23;135(6):e186388).

To clarify this point, the following text was added to the introduction to clarify the aim and importance of this study; ‘’Although ⁶⁸Ga-based radiopharmaceuticals are increasingly used, many conventional chelators still require heating, long preparation times, and post‑labeling purification, which limits routine clinical implementation. The use of the THP chelator offers a distinct advantage by enabling rapid, room‑temperature complexation of ⁶⁸Ga at neutral pH, producing high radiochemical yields without additional purification. This efficient labeling directly addresses a key translational gap: the need for radiotracers that can be prepared reliably and reproducibly in routine clinical settings, including sites without advanced radiochemistry infrastructure. In this manuscript, we build on these advantages of THP to demonstrate that THP‑conjugated Affibody molecules can facilitate diagnostic procedures, thereby providing a practical route toward more accessible and scalable clinical translation.’’

You are right about images. Overall biodistribution and particularly tumour uptake is similar for both radioconjugates. The important finding of this study is to achieve a similar biodistribution and also signal from the tumours using both Affibody-based radioconjugates while performing labeling more efficiently (quicker at room temperature with higher radiochemical yield and skipping the purification step) using THP-conjugated affibody molecules. Also, the following text was already included at the end of the Introduction: “This study compared the effects of substituting the macrocyclic chelator NOTA with the acyclic chelator THP on the biodistribution of 68Ga-labeled Affibody molecule for imaging B7-H3 expression. ”

2-The data show the systemic uptake of THP over NOTA in various organs, but there is no mechanistic explanation through experiments that have been explored. Additional in-vitro assays will strengthen the manuscript.

Answer. The uptake of a radiotracer in normal organs might happen because of its interaction with a molecular target expressed in normal tissues, as well as because of off-target interactions. The data from the Human Protein Atlas suggest that there is some B7-H3 protein expression in the cerebral cortex, colon, liver, testes, and lymph nodes. The murine B7-H3 is homologous to human, and ZB7-H3_2 binds to the murine B7-H3. The low accumulation of both [⁶⁸Ga]Ga-THP-ZB7-H3_2 and [⁶⁸Ga]Ga-NOTA-ZB7-H3_2 in normal organs compared to tumours suggests  that the true, on-target interaction plays a minor role in such uptake. The off-target interactions might be numerous, caused by a multitude of mechanisms, and often combined. Thus, it is difficult to model in vitro. A biodistribution experiment is always more informative to find if a particular modification results in undesirable changes in biodistribution.

3-The experiment with PET shows only one time point, which is insufficient to understand kinetics, tumor retention, etc. A time-activity graph is needed to determine if the slight uptake of THP resolves or worsens over time.

Answer. This time point was selected since the clinical studies demonstrated that HER2-targeting Affibody molecules provide high tumor uptake and high tumor-to-background ratio 2 h after injection. From a clinical point of view, we decided to perform imaging at 2h. Increasing the time between injection and imaging did not increase imaging contrast. However, further extension of the interval would result in the further decay of the short-lived ⁶⁸Ga.”

4-In the current version, only B7-H3-High and B7-H3-negative are used, which does not reflect the heterogeneity of its expression and TME. The author should include additional positive models to claim the heterogeneity.

Answer. We appreciate the reviewer’s insightful comment regarding the heterogeneity of B7‑H3 expression and the tumor microenvironment (TME). We fully agree that B7‑H3 expression varies across tumor types and even within individual tumors, and that the heterogeneity is an important consideration for clinical translation. In the present study, our primary objective was to evaluate the impact of chelator selection (THP vs. NOTA) on radiolabeling efficiency, biodistribution, and targeting properties of the same Affibody scaffold. To isolate the effect of the chelator and avoid confounding biological variables, we intentionally selected one B7‑H3‑high model (SKOV‑3) and one B7‑H3‑negative model (Ramos). This design allowed us to confirm specificity of the tracer in vivo, ensure that differences in biodistribution were attributable to the chelator rather than biological heterogeneity, and perform a controlled head‑to‑head comparison under identical experimental conditions. Additional tumor models would provide a broader biological context. However, such an expansion would require a substantially larger experimental framework, including characterization of B7‑H3 levels, optimization of dosing, and additional biodistribution and imaging studies. This is beyond the scope of the current methodological comparison, which focuses specifically on the translational advantages of THP‑based radiolabeling.

5-The author should mention the current ex vivo experimentation related to the B7-H3 receptor densities, along with old published data. Only providing the old data will not help to address how robustly THP reflects actual target expression.

Answer. Thank you for highlighting the importance of ex vivo experimentation related to B7‑H3 receptor density. In the revised manuscript, we now explicitly discuss how our current ex vivo biodistribution data relate to known receptor densities in the SKOV‑3 model. SKOV‑3 xenografts have been quantitatively characterized in our previous study (e.g., ref. 20), demonstrating high B7‑H3 expression. Our ex vivo uptake measurements therefore, reflect tracer performance in a model with a well-defined receptor abundance. We have added a paragraph to the Discussion clarifying that the comparable tumor uptake of THP‑ and NOTA‑based tracers indicates that THP labeling preserves the ability to report actual target expression. We also acknowledge that future work will include other models to broaden biological validation.

The following text was added to the discussion.

“In the present study, ex vivo biodistribution measurements provide a direct quantitative readout of tracer uptake in B7‑H3–expressing tumors, which indirectly reflects the available receptor density under our experimental conditions. The SKOV‑3 xenograft model used here has been characterized in previous studies, where B7‑H3 expression levels were quantified [20]. Our ex vivo data show that [⁶⁸Ga]Ga‑THP‑ZB7‑H3₂ achieves tumor uptake comparable to the NOTA‑based analogue despite differences in chelation chemistry, indicating that the THP‑based tracer robustly reports target expression in a model with well‑defined B7‑H3 abundance. Although we did not perform new receptor‑density quantification in this study, the combination of (i) established B7‑H3 expression levels in SKOV‑3 tumors, (ii) in vivo specificity confirmed by comparison with B7‑H3‑negative Ramos xenografts, and (iii) consistent ex vivo uptake patterns supports the conclusion that THP‑based labeling preserves the ability of the Affibody molecule to reflect B7‑H3 expression in vivo. ”

6-Provide full kinetics of binding strength along with IC50 and EC50, which limits the ability to determine if THP alters the interaction or not.

Answer. Thank you for pointing this out! In the current study, we quantified binding using EC₅₀ (cell‑based binding assay) and IC₅₀ (competition assay) to compare the apparent binding strength of THP‑ZB7‑H3₂ and NOTA‑ZB7‑H3₂. Both constructs showed identical EC₅₀ values and very similar IC₅₀ values, indicating that chelator substitution does not measurably affect apparent affinity under our experimental conditions. We agree that full kinetic parameters (kon, koff, KD) obtained by techniques such as SPR would provide additional mechanistic insight; however, such measurements were not performed and are beyond the scope of this work, which is primarily focused on radiolabeling efficiency, stability, and in vivo biodistribution. We have now explicitly acknowledged this limitation and clarified in the Discussion. ‘’ The combined EC₅₀ and IC₅₀ data, in vitro processing, and in vivo uptake strongly support the conclusion that THP does not significantly alter the interaction with B7‑H3.”

 

7-The paper mentioned Affibody as a reference and talks more about its safety, but the author fails to discuss whether the THP chelator and labeling condition initiate any risk either in animal study or experimental validations. Concluding the sentence below for THP conjugate affibody “These findings indicate that the THP-conjugated Affibody molecule is a promising candidate for future clinical translation.” would be strong, without providing any dosimetry recordings or comparison with B7-H3imaging.

Answer. Thank you for raising an interesting point for consideration! To address your comment, the following text was added to the Discussion: ‘’The use of THP does not introduce additional safety concerns under the conditions applied in this study. THP has been used in several preclinical [33, 35-38] and clinical [39,40] ⁶⁸Ga radiopharmaceuticals and has shown excellent in vivo stability and no adverse clinical effects at tracer-level doses. More than 100 patients have received THP-conjugated tracers in clinics, and no adverse clinical effects have been reported [39, 40]. Moreover, the radiolabeling procedure itself is performed at room temperature and neutral pH, conditions that do not alter the structure or integrity of the Affibody molecule. Consistent with this, no signs of toxicity or abnormal behavior were observed in any of the animals during biodistribution or imaging studies. Formal toxicology studies will provide more precise evidence regarding this point. However, the available evidence supports that THP conjugation and the associated labeling conditions do not pose additional risk in the experimental setting used here.

Clinical studies of ABY-025 show that the absorbed doses from affibody molecules are much lower than the dose limits set by toxicity risks.  On the other hand, the upscaling of dosimetry from mice to humans is associated with big uncertainties, and accuracy within an order of magnitude is quite common. For developing an imaging agent, the imaging contrast is much more important than the dosimetry.

We are currently developing a theranostic variant of the Affibody molecule, and comprehensive dosimetry studies for this construct to quantitatively assess absorbed doses and evaluate its suitability for both diagnostic imaging and targeted radionuclide therapy are underway.

We have revised the conclusion: ‘’The preliminary data indicate that the THP-conjugated Affibody molecule could be a promising candidate for future clinical translation’’.

Reviewer 3 Report

Comments and Suggestions for Authors

The THP-chelator is a promising tool for radiolabeling of Affibodies.

I am just missing logD values of the NOTA- and the THP-radioligand's. Could they be added please?

Author Response

Dear Reviewer,

We would like to sincerely thank you for the time and effort you devoted to reviewing our manuscript. We greatly appreciate your careful evaluation and constructive comments, which have been very helpful in improving the quality, clarity, and scientific rigor of our work.

Your insights and suggestions were carefully considered, and we believe they have significantly strengthened the manuscript. Below, we provide a point-by-point response to each of your comments.

Thank you again for your valuable input and contribution to the review process.

Kind regards,
Maryam Oroujeni, PhD

The THP-chelator is a promising tool for radiolabeling of Affibodies.

Answer. Thank you for the positive feedback on our papr!  

I am just missing logD values of the NOTA- and the THP-radioligand's. Could they be added please?

Answer. Thank you for pointing this out! We agree that logD measurements would provide additional quantitative information on the physicochemical differences between the two radioligands and could help interpret organ uptake patterns more mechanistically. However, the affibody molecules contain both amino acids with both hydrophobic and hydrophilic side chains. In an aqueous solution, the hydrophobic interaction between lipophilic amino acids provides a structural stability of the affibody scaffold, while the hydrophilic amino acids are exposed to water. However, when an affibody is exposed to n-octanol, the lipophilic size chains get exposed to the hydrophobic solvent. According to our previous experience, their interaction with n-octanol is predominant and masks more subtle differences in logD caused by the chelators. Thus, we cannot obtain informative data from this experiment for Affibody-based tracers.