Viral Reactivation in Multiple Myeloma Patients Receiving Anti-BCMA Chimeric Antigen Receptor T-Cell Therapy

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors analyzed viral reactivation in 75 patients treated with anti-BCMA CAR-T for multiple myeloma (MM) and compared them with 60 patients receiving commercial anti-CD19 CAR-T for B-cell lymphoma. They found that the majority of the CMV reactivations (87% and 68.5%, respectively) were asymptomatic and did not affect progression-free survival (PFS) or overall survival (OS) .

Highlights: This study characterizes the incidence and clinical impact of viral reactivation in patients with MM receiving anti-BCMA CAR-T cell therapy.

The manuscript is clinically significant and well organized. However, several issues remain to be addressed.

1. Please provide a citation for the statement “while EBV reactivation is observed in approximately 2% of cases.”in lines 54 - 55.
2. Please provide additional details on HBI0101 in line 78, particularly its targets and therapeutic rationale, for clarity.
3. Please clarify the meaning of "-" in the legends of Tables 1 and 2.
4. Please format “10^6”as a superscript (10⁶) in Table 3.
5. It is recommended to perform a multivariate analysis to examine the association between various CAR-T toxicities and CMV reactivation risk in patients with multiple myeloma .
6. Please provide details on the adjusted lymphodepletion regimen based on renal function in line 298.
7. It is advisable to perform statistical analysis using the new version of SPSS.
8. There are several mistakes in the References section. Please verify all the references and make sure that they are correct and consistent.
9. The manuscript contains several grammatical errors requiring correction. Please consult a native English speaker.
10. Please spell out the full form of each abbreviation upon first use.

Author Response

Dear Reviewer,

We would like to thank you for your thoughtful review of our manuscript: Viral Reactivation in Multiple Myeloma Patients Receiving Anti-BCMA
Chimeric Antigen Receptor T-Cell Therapy. We greatly appreciate the time and effort you dedicated to providing constructive feedback, which has significantly improved the clarity and quality of our paper.

We have carefully addressed all of the reviewers’ comments in the revised version. Below, please find our point-by-point responses to your comments

We thank you again for your time and consideration, and we hope that the revised manuscript is now suitable for publication in the journal.

Sincerely,
Eyal Lebel
Shlomo Elias

 

Comment 1: Please provide a citation for the statement “while EBV reactivation is observed in approximately 2% of cases.” in lines 54 - 55.

Response: We thank the reviewer for pointing this out. We have added the appropriate citation to support this statement in the revised manuscript (page 2, line 54-55).

 

Comment 2: Please provide additional details on HBI0101 in line 78, particularly its targets and therapeutic rationale, for clarity.

Response: We appreciate the suggestion to clarify this point. HBI0101 is a novel, second-generation, optimized anti-BCMA CAR-T product developed as a point-of-care treatment. Its development aims to bypass current economic and logistical constraints to ensure broader patient access to potent therapies. We have expanded the description of its target and therapeutic rationale in the revised manuscript (page 2, line 78-81).

Comment 3: Please clarify the meaning of "-" in the legends of Tables 1 and 2.

Response: Thank you for highlighting this ambiguity. The "-" symbol was originally intended to designate "not applicable" values or tests. To improve clarity, we have replaced all instances of "-" with "N/A" in Tables 1 and 2, and we have added an explanatory note to the table legends to reflect this change.

 

Comment 4: Please format “10^6” as a superscript (106) in Table 3.

Response: The text in Table 3 has been corrected to properly display 10^6 with a superscript.

 

Comment 5: It is recommended to perform a multivariate analysis to examine the association between various CAR-T toxicities and CMV reactivation risk in patients with multiple myeloma.

Response:

We thank the reviewer for raising this important point. As shown in Tables 3 and 4, univariate analyses were performed to evaluate the associations between CMV reactivation and pre-infusion factors as well as CAR-T–related toxicities. In each analysis, only a single variable reached statistical significance. Accordingly, a multivariable model was not initially pursued. In response to the reviewer’s suggestion, we have now performed a multivariable analysis including all evaluated parameters. The results remained unchanged, with the same variables retaining statistical significance.

 

Comment 6: Please provide details on the adjusted lymphodepletion regimen based on renal function in line 298.

Response: Patients with a creatinine clearance of less than 30 mL/min received bendamustine (90 mg/m2 for 2 days) instead of the standard fludarabine and cyclophosphamide regimen. We have added these specific regimen details to the revised manuscript (page 10, line 314-315).

 

Comment 7: It is advisable to perform statistical analysis using the new version of SPSS.

Response: We thank the reviewer for this observation. We would like to clarify that the statistical analyses were performed using the most current version of SPSS (version 31.0.0). The reference to an earlier version in the original submission was an error, which has now been corrected in the revised manuscript (page 11, line 355).

Comment 8: There are several mistakes in the References section. Please verify all the references and make sure that they are correct and consistent.

Response: We thank the reviewer for this careful observation. Following the reviewer’s comment, we have carefully reviewed and corrected the References to ensure all citations are accurate and consistent with the journal's guidelines.

 

Comment 9: The manuscript contains several grammatical errors requiring correction. Please consult a native English speaker.

Response: We thank the reviewer for this feedback. The new version of the manuscript has undergone a thorough proofreading and editing process to correct grammatical errors and improve the overall language and flow of the text.

 

Comment 10: Please spell out the full form of each abbreviation upon first use.

Response: Thank you for pointing this out. We have carefully reviewed the text to ensure that every abbreviation is fully defined upon its first appearance in the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript investigates the incidence and clinical impact of viral infections in multiple myeloma (MM) patients following anti-BCMA CAR-T cell therapy. The authors report that although CMV and EBV reactivations are relatively common, they are rarely associated with clinically meaningful disease, suggesting that routine pre-emptive viral screening in asymptomatic patients may be unnecessary. Overall, this is an interesting and timely study that addresses an important clinical concern in BCMA CAR-T therapy, and the data are clearly presented.

However, the manuscript would be strengthened by addressing the following point: the timing of viral reactivation relative to BCMA CAR-T cell infusion is not clearly described. Specifically, it is unclear how long after CAR-T treatment viral reactivation was detected in affected patients.

Author Response

Dear Reviewer,

We would like to thank you for your thoughtful review of our manuscript: Viral Reactivation in Multiple Myeloma Patients Receiving Anti-BCMA
Chimeric Antigen Receptor T-Cell Therapy. We greatly appreciate the time and effort you dedicated to providing constructive feedback, which has significantly improved the clarity and quality of our paper.

Below, please find our point-by-point responses to your comments

We thank you again for your time and consideration, and we hope that the revised manuscript is now suitable for publication in the journal.

Sincerely,
Eyal Lebel
Shlomo Elias

 

Comment 1: The timing of viral reactivation relative to BCMA CAR-T cell infusion is not clearly described. Specifically, it is unclear how long after CAR-T treatment viral reactivation was detected in affected patients.

Response: We thank the reviewer for highlighting this important point, which required further clarification. In the multiple myeloma (MM) cohort, the mean and median times to CMV reactivation (rCMV) were 176.8 and 70 days, respectively, compared with 241.1 and 96 days in the B-cell lymphoma (BCL) cohort (p=1.00 and p=0.53, respectively). The time to rCMV ranged from 10 to 769 days in the MM cohort, and from 7 to 928 days in the BCL cohort. We have added a detailed description of the timing of viral reactivation relative to the CAR-T cell infusion in the revised manuscript (page 4, line 115-119).

Reviewer 3 Report

Comments and Suggestions for Authors

This paper is focusing on very important issue inside novel cell therapies. Infections are a most common complication of CAR-T cell treatment. The paper is well written and has a clear message for the question of virus load/viremia.

I have some small comment to authors:

Abbreviation "CS-rCMV" is explained very late in the paper, on row  195, should be opened when mentioned first time.

Page 4 Table 1: there are many parameters and showed as like  "IgG pre-inf Mean +/- SD"

like IgG 1271 +/- 1661, how is 1271-1661 possible? Should all these parameters be other way round?

Regarding PCR tests, please clarify what days +7-14 mean; every day between these days or once between these days?

Higher ferritin levels seemed to associate with the risk of virus activation. What is the opinion of authors; would it be beneficial to test patients with higher ferritin to diagnose rising levels of virus on time?

Same concerns the patient with progressive disease who seemed to be at risk of rCMV as well. I am wondering should we have recommendation to test those patients frequently if not all CART cell patients?  At least those with BiTes. Those patients are in danger if rCMV activation occurs and treament of progressive disease must be delayed.

The authors declare there is no funding, however, in Acknowledgements several funds, awars and grants are mentioned. Should these move to the paragraph of funding.

 

Author Response

Dear Reviewer,

We would like to thank you for your thoughtful review of our manuscript: Viral Reactivation in Multiple Myeloma Patients Receiving Anti-BCMA
Chimeric Antigen Receptor T-Cell Therapy. We greatly appreciate the time and effort you dedicated to providing constructive feedback, which has significantly improved the clarity and quality of our paper.

Below, please find our point-by-point responses to your comments

We thank you again for your time and consideration, and we hope that the revised manuscript is now suitable for publication in the journal.

Sincerely,
Eyal Lebel
Shlomo Elias

 

Comment 1: Abbreviation "CS-rCMV" is explained very late in the paper, on row 195, should be opened when mentioned first time.

Response: Thank you for pointing this out. We have updated the manuscript to ensure the abbreviation "CS-rCMV" is fully defined upon its first appearance in the text (page 4, line 118).

 

Comment 2: Page 4 Table 1: there are many parameters and showed as like "IgG pre-inf Mean +/- SD" like IgG 1271 +/- 1661, how is 1271-1661 possible? Should all these parameters be other way round?

Response: We thank the reviewer for this careful observation. The values presented in Table 1 are indeed correct as written and have not been reversed. The large SD reflects a wide variance, with a right-skewed distribution driven by a subset of patients with markedly elevated IgG levels, rather than a normal distribution.

 

Comment 3: Regarding PCR tests, please clarify what days +7-14 mean; every day between these days or once between these days?

Response: We thank the reviewer for pointing out this ambiguity. Our intent was that the PCR test was performed once within the timeframe of days +7 to +14, rather than daily. We have updated the manuscript accordingly to ensure this schedule is clearly stated (page 11, line 340)

 

Comment 4: Higher ferritin levels seemed to associate with the risk of virus activation. What is the opinion of authors; would it be beneficial to test patients with higher ferritin to diagnose rising levels of virus on time?

Response: We appreciate the reviewer’s insightful question. We agree that elevated ferritin levels have the potential to serve as an important predictive biomarker for identifying patients at high risk of CMV reactivation. Ferritin is already routinely measured in patients undergoing CAR-T therapy, as it contributes to the hematotox score and helps assess the risk of hematotoxicity following CAR-T. However, while our current findings suggest this association, this relationship requires further investigation and validation in larger, prospective cohorts before recommending routine, targeted viral monitoring based on baseline ferritin levels. We now address this point in the revised manuscript (page 9, line 275).

 

Comment 5: Same concerns the patient with progressive disease who seemed to be at risk of rCMV as well. I am wondering should we have recommendation to test those patients frequently if not all CART cell patients? At least those with BiTes. Those patients are in danger if rCMV activation occurs and treament of progressive disease must be delayed.

Response: We strongly agree with the reviewer's clinical insight. While establishing formal guidelines for routine screening across all CAR-T or BiTE recipients requires further prospective validation, heightened vigilance is certainly warranted. For patients exhibiting the risk factors identified in our study, or those recognized in the broader literature, targeted CMV monitoring following disease progression could be highly beneficial. Those with progressive disease or other identified risk factors may benefit from targeted CMV monitoring to prevent treatment delays in subsequent therapies.  We have added a discussion of this point in the revised manuscript (page 10, line 283-290).

 

Comment 6: The authors declare there is no funding, however, in Acknowledgements several funds, awars and grants are mentioned. Should these move to the paragraph of funding.

Response: We thank the reviewer for allowing us to clarify this point. There was indeed no direct funding received for this specific study. The various grants and awards listed in the Acknowledgements were awarded to the researchers in support of different, separate research. They were included solely for transparency and general disclosure, not as direct funding for the present work. The issue was clarified in the revised manuscript (page 11, line 381).

Reviewer 4 Report

Comments and Suggestions for Authors

In this article, Cohen et al. carried out a study tracking the viral reactivation rate and their clinical significance in multiple myeloma (MM) patients receiving anti B-Cell Maturation Antigen (BMCA) Chimeric Antigen Receptor T (CAR-T) cell therapies. Reactivation of viruses, such as Cytomegalovirus (CMV) or Epstein-Barr Virus (EBV) can result in significant clinical consequences while utilizing CAR-T therapies, especially for MM patients. Authors compared incidence and clinical consequences of CMV, EBV and Adenovirus (AV) reactivations in MM patients treated with anti-BMCA CAR-T therapies and compared them against B-Cell Lymphoma patients treated with anti-CD19 CAR-T therapies. Some CMV and EBV reactivations were observed across both cohorts, but AV reactivation was not detected. Additionally, most viral reactivations were asymptomatic and clinically insignificant.

 

Overall, this is a concisely written manuscript that makes intriguing observations into frequency of viral reactivations and their clinical consequences in MM patients treated with BMCA CAR-T therapies. Conclusions are well supported by the data presented in the manuscript, although as authors point out, data were derived from a small cohort of patients at a single clinical center. Still, I believe this manuscript makes observations that can be useful to the hematologic malignancies and CAR-T therapy fields.

 

I think the manuscript is well written with sufficient details and is mostly good to go. I have a small suggestion that may help improve the manuscript.

 

1. In table 2, authors show blood CMV viral load (copies/mL) for MM and BCL patients that exhibited clinical symptoms following viral reactivations. I am curious what these numbers looked like for asymptomatic patients? Authors show the median value (695 copies / mL) in MM patients, but I’m curious if there were any patients with high viral loads and remained asymptomatic. Would it be possible for the authors to showcase all of the data points for viral loads and whether they exhibited symptoms with a dotplot?

Author Response

Dear Reviewer,

We would like to thank you for your thoughtful review of our manuscript: Viral Reactivation in Multiple Myeloma Patients Receiving Anti-BCMA
Chimeric Antigen Receptor T-Cell Therapy. We greatly appreciate the time and effort you dedicated to providing constructive feedback, which has significantly improved the clarity and quality of our paper.

In response to the reviewers’ suggestions, we have also added an additional figure, which is now included in the supplementory material.

Below, please find our point-by-point responses to your comments

We thank you again for your time and consideration, and we hope that the revised manuscript is now suitable for publication in the journal.

Sincerely,
Eyal Lebel
Shlomo Elias

 

Comment 1: In table 2, authors show blood CMV viral load (copies/mL) for MM and BCL patients that exhibited clinical symptoms following viral reactivations. I am curious what these numbers looked like for asymptomatic patients? Authors show the median value (695 copies / mL) in MM patients, but I’m curious if there were any patients with high viral loads and remained asymptomatic. Would it be possible for the authors to showcase all of the data points for viral loads and whether they exhibited symptoms with a dotplot?

Response: We thank the reviewer for this excellent suggestion. To provide a clearer picture of the viral load distribution across both symptomatic and asymptomatic patients, we have generated the requested dot plot, now included in the Supplementary Material (Supplementary Figure 1). The figure displays all individual data points for viral loads in both the MM and BCL cohorts, stratified by the presence or absence of clinical symptoms.

As illustrated, a subset of patients exhibited high viral loads while remaining entirely asymptomatic, yet the overall median viral load was lower in asymptomatic patients

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

We appreciate the authors' detailed revision. However, some points still require resolution.

1. As suggested in the first review, the authors have added a citation for the statement “while EBV reactivation is observed in approximately 2% of cases.”in lines 54 - 55. However, this citation does not mention anything about EBV reactivation.  
2. Several grammatical errors, including missing predicates and singular-plural mismatches, remain in the revised manuscript. Professional language editing by a native English speaker or a certified scientific editing service is recommended.

Author Response

Dear reviewer,
We would like to thank you again for your review of our manuscript

Comment 1: As suggested in the first review, the authors have added a citation for the statement “while EBV reactivation is observed in approximately 2% of cases.” in lines 54 - 55. However, this citation does not mention anything about EBV reactivation.
Response: The incorrect citation has been replaced.

Comment 2: Several grammatical errors, including missing predicates and singular-plural mismatches, remain in the revised manuscript. Professional language editing by a native English speaker or a certified scientific editing service is recommended.
Response: The manuscript has now been reviewed and corrected by a professional scientific language editor to resolve the remaining grammatical issues.

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

As stated in the cited document, “Four patients had EBV reactivation after infusion, which were all asymptomatic.” Please recalculate EBV reactivation rate in reference 6. In addition, reference 6 concerns anti-BCMA CAR-T therapy, rather than anti-CD19 CAR-T therapy mentioned in lines 51-53.

Author Response

We thank the reviewer for this helpful comment. We recalculated the EBV reactivation rate reported in reference 6 based on the four documented cases and revised the text accordingly. We also clarified that reference 6 concerns anti-BCMA CAR-T therapy rather than anti-CD19 CAR-T therapy, and adjusted the wording to distinguish between these studies (lines 51-64).  The text of the manuscript was proofread by a professional English-language editor.

Round 4

Reviewer 1 Report

Comments and Suggestions for Authors

I have no further comments.