Application of Engineered NK-92 Cell Extracellular Vesicles in the Treatment of Systemic Lupus Erythematosus

Reviewer #1 (Public review):

Summary:

This study constructed engineered NK-92 cell extracellular vesicles displaying CD19 single-chain variable fragment and evaluated their therapeutic efficacy in MRL/lpr mouse models of systemic lupus erythematosus, demonstrating that these vesicles could deplete B cells, alleviate lupus nephritis, and improve mouse survival. However, this strategy lacks significant innovation compared to existing research. The current results are not sufficient to provide strong support for the experimental hypotheses.

Weaknesses:

(1) This study proposes using engineered EVs displaying CD19 scFv to target B cells for SLE treatment. However, similar core therapeutic strategies have been reported in previous studies. For instance, recently, studies have reported engineered EVs for SLE therapy (J Control Release. 2025, 384:113886; Ann Rheum Dis. 2025, 84(11):1811-1821; J Nanobiotechnology. 2026, 24(1):203). Another research team from China also constructed engineered EVs displaying anti-CD19 scFv for SLE treatment, which is highly consistent with the present work in targeting strategy, delivery vehicle, and disease model (Mol Ther. 2026:S1525-0016(26)00080-8). Moreover, the human trial of allogeneic CD19-targeted CAR-NK therapy for SLE has been published (Lancet. 2026, 406(10522):2968-2979). This study has not made original improvements in therapeutic vectors, targeting modules, therapeutic mechanisms, and indications, and thus finds it difficult to meet the requirements of high-level journals for originality and novelty.

(2) Numerous core experiments are missing, including the validation of CD19 scFv fusion protein expression on EVs, systematic characterization of engineered EVs, verification of EVs functions and therapeutic mechanisms, and in vitro and in vivo safety assessments. The available data are insufficient to support complete conclusions.

(3) The stable expression of CD19 scFv on EVs should be further verified by Western blot or flow cytometry. The anchoring of CD19 scFv on the outer membrane surface of EVs must be confirmed. In addition, the loading capacity of CD19 scFv on exosomes should be quantified for the dosage selection in SLE treatment.

(4) In vitro experiments are required to confirm the specific targeting ability of CD19 scFv-EVs to B cells and clarify the precise mechanism of B cell depletion, particularly whether it is mediated by effector molecules carried by exosomes such as perforin and granzyme B.

(5) The key quality control parameters, such as the stability, purity, buoyant density, and particle/protein ratio of engineered exosomes, should be characterized and identified.

(6) For the in vivo treatment experiments, the author needs to explain how the treatment dose of CD19scFv-EVs was determined in order to clarify the dose-effect relationship.

(7) It is necessary to supplement with in vivo imaging and tissue distribution data to prove that the CD19 scFv-EVs can specifically accumulate in B-cell organs such as the spleen or lymph nodes.

(8) The author needs to clarify the mechanism by which CD19 scFv-EVs reduce B cells in vivo and verify the caspase apoptosis pathway.

(9) For the in vivo therapeutic experiments, the clinical first-line drugs and the free CD19scFv should be used to supplement the control group to highlight the advantages of the engineered EVs.

(10) Safety assessment in this manuscript is completely absent. Routine toxicity examinations, including hepatic and renal function tests, routine blood tests, and histopathological analysis of major organs in mice, must be supplemented. In addition, the systemic inflammatory cytokine profile and anti-drug antibody levels should be determined to rule out critical safety risks such as cytokine release syndrome and immunogenicity. The authors only focused on alterations in B cells; the impacts of the treatment on T cell subsets, NK cells, and monocytes/macrophages should be further investigated.

Reviewer #2 (Public review):

Summary:

Sun and colleagues report the development of an engineered extracellular vesicle platform derived from NK-92 cells that display an anti-CD19 single-chain variable fragment (scFv) on their surface via fusion with LAMP-2B (V-CD19-Exo). In an MRL/lpr mouse model of SLE, the authors demonstrate that intraperitoneal administration of V-CD19-Exo reduces splenic CD19+CD20+ B cells, attenuates proteinuria and lupus nephritis pathology, downregulates pro-inflammatory cytokines (IL-17A, IFN-γ) and autoantibodies (anti-dsDNA, ANA), and improves survival from approximately 25% to 80%. The authors propose that this "cell-free" targeted extracellular vesicle strategy offers advantages over conventional cell therapies, including lower immunogenicity, scalable production, and no requirement for lymphodepletion.

The study addresses an important question in autoimmune disease therapeutics: how to achieve targeted B cell depletion while avoiding the complexities and safety risks associated with CAR-T/CAR-NK cell therapies. The concept is novel, and the initial in vivo efficacy data are encouraging. However, several significant limitations in experimental design, mechanistic depth, and evidence rigor temper the strength of the conclusions.

Strengths:

(1) Novel conceptual approach.

The adaptation of CAR targeting principles to extracellular vesicles represents a creative and potentially impactful strategy. By displaying CD19 scFv on NK-92-derived vesicles, the authors successfully confer B cell-targeting capability while retaining the cytotoxic effector functions of the parental NK cells. This "cell-free" concept addresses genuine limitations of live cell therapies, including the need for lymphodepletion, risks of cytokine release syndrome, and manufacturing complexity.

(2) Comprehensive in vivo efficacy readouts.

The study evaluates therapeutic effects across multiple clinically relevant endpoints: B cell depletion (flow cytometry), renal function (proteinuria, UPCR), renal histopathology (HE staining with semi-quantitative scoring), systemic inflammation (IgE, IL-17A, IFN-γ), autoantibody production (anti-dsDNA, ANA), and survival. This multi-dimensional characterization strengthens the phenotypic evidence for efficacy.

(3) Appropriate control groups.

The inclusion of non-targeted NK92-Exo as a control allows attribution of the observed effects to CD19-mediated targeting rather than non-specific vesicle-associated activities.

(4) Significant survival benefit.

The improvement in survival from 25% to approximately 80% in V-CD19-Exo-treated mice is substantial and represents arguably the most compelling evidence for therapeutic potential in this model.

Weaknesses:

(1) Mechanism of B-cell reduction remains unclear.

The manuscript reports a dramatic reduction in splenic CD19+CD20+ B cells (from 10.53% to 1.51%) following V-CD19-Exo treatment. However, the authors do not establish whether this results from direct cytotoxicity (e.g., perforin/granzyme-mediated killing, apoptosis induction) or from functional suppression/downregulation of CD19 expression. The authors speculate that the effect is likely mediated by cytotoxic proteins carried by NK-92-derived vesicles, but no data are provided to support this mechanism. Essential experiments would include the detection of apoptosis markers (Annexin V, activated caspase-3/7) in B cells, assessment of perforin/granzyme B content within V-CD19-Exo, or in vitro co-culture assays demonstrating direct B cell killing.

(2) Small sample sizes.

Most experimental endpoints were assessed with n=5 per group, which is marginal for detecting modest effect sizes and may amplify the influence of individual biological variation. While the survival study had n=10 per group, the main mechanistic and endpoint analyses would benefit from larger cohorts (n=8-10) to increase statistical power and robustness.

(3) No dose-response or dosing optimization studies.

All experiments used a single dose (10⁹ particles per injection) and a fixed schedule (twice weekly for three weeks). The absence of dose-response data leaves unclear whether the observed effects represent maximal efficacy or could be achieved with lower doses, and whether alternative dosing regimens could improve outcomes or reduce potential off-target effects.

(4) Lack of safety assessment.

The authors emphasize the theoretical safety advantages of extracellular vesicles over cell therapies, but no systematic safety evaluation is presented. Key missing data include: histopathological examination of non-target organs (liver, lung, heart, gastrointestinal tract), assessment of off-target immune activation (T cell responses, cytokine profiles beyond those measured), and evaluation of potential accumulation or toxicity with repeated dosing.

(5) Incomplete characterization of the engineered vesicles beyond targeting.

While the manuscript successfully demonstrates CD19scFv display and vesicle enrichment of exosomal markers, it does not characterize whether V-CD19-Exo retains the full spectrum of NK-92 effector molecules (perforin, granzymes, FasL, TRAIL, cytokines such as IFN-γ) at functional levels. Quantitative or semi-quantitative comparison of cargo between V-CD19-Exo and parental NK-92 cells or non-engineered NK92-Exo would help contextualize the observed in vivo effects.

(6) Sex as a biological variable is not systematically addressed.

The authors note in the Discussion that the same treatment showed more significant efficacy in male mice compared to females (data not shown), yet all main experiments were conducted exclusively in female mice. Given the strong sex bias in SLE epidemiology (approximately 9:1 female-to-male ratio) and potential differences in immune responses between sexes, this observation warrants systematic investigation rather than a footnote. Presenting the sex-differential data or alternatively, conducting adequately powered sex-stratified analyses would substantially strengthen the manuscript.

(7) Translational claims are premature.

The manuscript repeatedly emphasizes advantages over cell therapy (low immunogenicity, scalable production, no requirement for lymphodepletion) as if these are established properties of V-CD19-Exo. However, no experiments directly compare V-CD19-Exo to CAR-NK or CAR-T cells in terms of efficacy, immunogenicity, or safety. Similarly, claims of "scalable production" and "high batch-to-batch consistency" are not supported by any manufacturing or quality control data. These statements should be toned down or supported with empirical evidence.

Reviewer #3 (Public review):

Summary:

This manuscript describes the development of engineered NK-92-derived extracellular vesicles (EVs) displaying CD19scFv for targeted treatment of systemic lupus erythematosus (SLE). Using a CD19scFv-LAMP2B fusion strategy, the authors generated EVs intended to selectively target pathogenic B cells in the MRL/lpr lupus mouse model. The study reports reductions in CD19⁺CD20⁺ B-cell populations, improvements in proteinuria and renal histopathology, decreased inflammatory cytokines and autoantibody levels, reduced splenomegaly, and improved survival outcomes following treatment. The work aims to position engineered EVs as a cell-free alternative to CAR-T/CAR-NK therapies for autoimmune disease treatment. While the concept is interesting and potentially translational, the study currently lacks sufficient methodological rigor, EV purification standards, mechanistic validation, and comprehensive characterization to fully support many of the claims presented.

Strengths:

(1) The study addresses an important unmet clinical need in systemic lupus erythematosus and explores an innovative cell-free therapeutic strategy.

(2) The concept of combining CAR-like targeting approaches with engineered EVs is interesting and potentially translational.

(3) The manuscript includes both in vitro and in vivo experiments, including functional renal assessments, immune profiling, histopathology, and survival studies.

(4) The authors attempt to evaluate multiple disease-associated readouts, including proteinuria, cytokines, autoantibodies, splenomegaly, and survival outcomes, which strengthens the overall biological relevance of the work.

(5) The use of engineered NK92-derived vesicles as a scalable alternative to CAR-NK therapy represents a potentially attractive therapeutic platform.

(6) The in vivo therapeutic observations in the MRL/lpr lupus model are encouraging and warrant further mechanistic investigation.

Weaknesses:

(1) The EV isolation strategy is not sufficiently rigorous for defining the isolated particles as "exosomes" according to current International Society for Extracellular Vesicles/MISEV guidelines. The precipitation-based workflow without density gradient purification or SEC raises major concerns regarding EV purity and identity.

(2) No direct validation was provided demonstrating successful surface localization or functional accessibility of CD19scFv on EV membranes.

(3) The characterization of EVs is incomplete and insufficient. Additional positive/negative EV markers, purity metrics, and orthogonal characterization methods are required.

(4) The absence of density gradient ultracentrifugation is particularly concerning, given the systemic injection of EV preparations into mice, as contaminating soluble factors and non-vesicular particles may contribute to the observed therapeutic effects.

(5) The manuscript lacks adequate mechanistic studies explaining how engineered EVs mediate B-cell depletion or immune modulation.

(6) The in vitro functional assays are weakly designed, particularly the use of A549 cells for evaluating CD19-targeted vesicle function.

(7) Important methodological details are missing, including EV normalization strategies, flow cytometry gating controls, blinding procedures, and randomization approaches.

(8) Several figures, particularly TEM and western blot images, are of low quality and difficult to interpret.

(9) The study does not sufficiently exclude the possibility that observed therapeutic effects result from contaminating soluble immune mediators rather than EV-specific activity.

(10) Broader immune profiling is lacking despite the systemic immune complexity of SLE.

(11) The statistical analysis section includes tests that are not reflected in the Results section, creating concerns regarding data presentation and consistency.

(12) Overall, while the concept is interesting, the manuscript currently falls short of the experimental rigor expected for high-impact translational EV studies.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

This study constructed engineered NK-92 cell extracellular vesicles displaying CD19 single-chain variable fragment and evaluated their therapeutic efficacy in MRL/lpr mouse models of systemic lupus erythematosus, demonstrating that these vesicles could deplete B cells, alleviate lupus nephritis, and improve mouse survival. However, this strategy lacks significant innovation compared to existing research. The current results are not sufficient to provide strong support for the experimental hypotheses.

Weaknesses:

(1) This study proposes using engineered EVs displaying CD19 scFv to target B cells for SLE treatment. However, similar core therapeutic strategies have been reported in previous studies. For instance, recently, studies have reported engineered EVs for SLE therapy (J Control Release. 2025, 384:113886; Ann Rheum Dis. 2025, 84(11):1811-1821; J Nanobiotechnology. 2026, 24(1):203). Another research team from China also constructed engineered EVs displaying anti-CD19 scFv for SLE treatment, which is highly consistent with the present work in targeting strategy, delivery vehicle, and disease model (Mol Ther. 2026:S1525-0016(26)00080-8). Moreover, the human trial of allogeneic CD19-targeted CAR-NK therapy for SLE has been published (Lancet. 2026, 406(10522):2968-2979). This study has not made original improvements in therapeutic vectors, targeting modules, therapeutic mechanisms, and indications, and thus finds it difficult to meet the requirements of high-level journals for originality and novelty.

J Control Release. 2025, 384:113886; Ann Rheum Dis. 2025, 84(11):1811-1821; J Nanobiotechnology. 2026, 24(1):203). Another research team from China also constructed engineered EVs displaying anti-CD19 scFv for SLE treatment, which is highly consistent with the present work in targeting strategy, delivery vehicle, and disease model (Mol Ther. 2026:S1525-0016(26)00080-8). Moreover, the human trial of allogeneic CD19-targeted CAR-NK therapy for SLE has been published (Lancet. 2026, 406(10522):2968-2979).

Reviewer 1 mentioned 4 publications

(1) J Control Release. 2025, 384:113886; Genetically engineered extracellular vesicles expressing decoy protein TACI provide a therapeutic effect in systemic lupus erythematosus mouse model

(2) Ann Rheum Dis. 2025, 84(11):1811-1821; J Nanobiotechnology. 2026, 24(1):203）Genetically modified CD19-targeting IL-15 secreting NK cells for the treatment of systemic lupus erythematosus. –but not Evs

(3) Lancet. 2026, 406(10522):2968-2979） Efficacy and safety of allogeneic CD19 CAR NK-cell therapy in systemic lupus erythematosus: a case series in China。

(4) Anti-CD19 engineered exosomes enable B-cell targeted anti-BAFF mRNA delivery to alleviate lupus progression”, 

We sincerely thank the reviewers for their valuable and constructive feedback. We fully acknowledge the important contributions made by the publications cited, and we respectfully submit that they do not invalidate our findings. A critical point to emphasize is that our study employed engineered NK-92 cell extracellular vesicles (EVs) not the cells themselves and we would like to respectfully reiterate the fundamental differences between whole cells and non-cellular EVs, particularly in terms of safety and efficiency profiles. Our safety hypothesis is further supported by the clinical use of inactivated NK-92 cells (as demonstrated in this study: [URL]), which we believe provides a strong and relevant precedent. We are also very grateful that the originality and novelty of our approach have been favorably recognized by Reviewers 2 and 3, which we take as an encouraging validation of our work.

(2) Numerous core experiments are missing, including the validation of CD19 scFv fusion protein expression on EVs, systematic characterization of engineered EVs, verification of EVs functions and therapeutic mechanisms, and in vitro and in vivo safety assessments. The available data are insufficient to support complete conclusions.

(3) The stable expression of CD19 scFv on EVs should be further verified by Western blot or flow cytometry. The anchoring of CD19 scFv on the outer membrane surface of EVs must be confirmed. In addition, the loading capacity of CD19 scFv on exosomes should be quantified for the dosage selection in SLE treatment.

We sincerely thank the reviewers for raising these important points. We note that points (2) and (3) address essentially the same concern, and we fully agree that further validation of CD19 scFv fusion protein expression on EVs is necessary. We are pleased to confirm that we will present additional data on this in due course. Furthermore, we respectfully acknowledge that several other aspects—including the EVs' functions, therapeutic mechanisms, in vitro and in vivo safety profiles, and CD19 scFv loading capacity—remain to be thoroughly investigated. We are committed to addressing these important questions in our follow-up studies, and we hope to provide more comprehensive insights in future work.

(4) In vitro experiments are required to confirm the specific targeting ability of CD19 scFv-EVs to B cells and clarify the precise mechanism of B cell depletion, particularly whether it is mediated by effector molecules carried by exosomes such as perforin and granzyme B.

We are most grateful to the reviewer for raising this important point. We are happy to report that we have successfully obtained data demonstrating the specific targeting of CD19 scFv-EVs to B cells, and we will be pleased to include these findings in our revision. With regard to the mechanism of action, we respectfully acknowledge that perforin and granzyme B are recognized as key mediators of NK cell targeting. Nevertheless, we are not aware of any published evidence to date that supports the presence of this same machinery in NK exosomes. We consider this a valuable question for future exploration, and while it lies beyond the scope of the current work, we are diligently investigating it in related ongoing studies.

(5) The key quality control parameters, such as the stability, purity, buoyant density, and particle/protein ratio of engineered exosomes, should be characterized and identified.

Agreed, We will provide additional characterization data for the engineered EVs in our revision.

(6) For the in vivo treatment experiments, the author needs to explain how the treatment dose of CD19scFv-EVs was determined in order to clarify the dose-effect relationship.

We sincerely thank the reviewer for this valuable suggestion. We fully agree and will be happy to revise the dose calculation accordingly in the updated manuscript.

(7) It is necessary to supplement with in vivo imaging and tissue distribution data to prove that the CD19 scFv-EVs can specifically accumulate in B-cell organs such as the spleen or lymph nodes. 

We sincerely thank the reviewer for this valuable suggestion. We fully acknowledge that this is a challenging experiment for several reasons: (1) EV internalization is a rapid process and is therefore difficult to capture; and (2) currently, there is no reliable method available for labeling EVs. Nevertheless, we respectfully assure the reviewer that we will make every effort to attempt this experiment and will report our findings in due course.

(8) The author needs to clarify the mechanism by which CD19 scFv-EVs reduce B cells in vivo and verify the caspase apoptosis pathway.

We sincerely thank the reviewer for these valuable comments. We are pleased to confirm that we have successfully demonstrated the specific targeting ability of CD19 scFv-EVs to B cells, and we will gladly incorporate these results in our revised manuscript.

Regarding the mechanism of action, we fully acknowledge that perforin and granzyme B are well-established mediators of NK cell targeting according to textbook knowledge. However, to the best of our knowledge, there is currently no evidence indicating that NK-derived exosomes are equipped with the same machinery. We respectfully recognize that this is an interesting and important question; while it lies beyond the scope of the present study, we are actively pursuing it in our ongoing parallel work.

We also appreciate the reviewer's comment regarding the apoptosis pathway. We respectfully note that this aspect was not assessed in any of the publications mentioned by Reviewer 1, which suggests that such analysis may be considered optional rather than mandatory. Nevertheless, we fully agree that this is a worthwhile avenue for further investigation, and we are committed to exploring it in our future studies."

(9) For the in vivo therapeutic experiments, the clinical first-line drugs and the free CD19scFv should be used to supplement the control group to highlight the advantages of the engineered EVs.

We sincerely thank the reviewer for this thoughtful and constructive advice. We fully agree that if we were developing this approach for clinical trials, regulatory agencies such as the FDA would require it to demonstrate superiority over current first-line clinical drugs. However, we respectfully wish to clarify that the primary objective of the present study is to provide a proof-of-concept that this strategy is feasible. We fully acknowledge that efficacy and safety will need to be investigated more intensively in future studies before any clinical translation can be considered. We are grateful for this valuable perspective and will be sure to discuss these considerations more explicitly in the revised manuscript.

(10) Safety assessment in this manuscript is completely absent. Routine toxicity examinations, including hepatic and renal function tests, routine blood tests, and histopathological analysis of major organs in mice, must be supplemented. In addition, the systemic inflammatory cytokine profile and anti-drug antibody levels should be determined to rule out critical safety risks such as cytokine release syndrome and immunogenicity. The authors only focused on alterations in B cells; the impacts of the treatment on T cell subsets, NK cells, and monocytes/macrophages should be further investigated.

We sincerely thank the reviewer for this valuable advice. We fully agree and will be happy to provide additional data to address this point in our revised manuscript.

Reviewer #2 (Public review):

Summary:

Sun and colleagues report the development of an engineered extracellular vesicle platform derived from NK-92 cells that display an anti-CD19 single-chain variable fragment (scFv) on their surface via fusion with LAMP-2B (V-CD19-Exo). In an MRL/lpr mouse model of SLE, the authors demonstrate that intraperitoneal administration of V-CD19-Exo reduces splenic CD19+CD20+ B cells, attenuates proteinuria and lupus nephritis pathology, downregulates pro-inflammatory cytokines (IL-17A, IFN-γ) and autoantibodies (anti-dsDNA, ANA), and improves survival from approximately 25% to 80%. The authors propose that this "cell-free" targeted extracellular vesicle strategy offers advantages over conventional cell therapies, including lower immunogenicity, scalable production, and no requirement for lymphodepletion.

The study addresses an important question in autoimmune disease therapeutics: how to achieve targeted B cell depletion while avoiding the complexities and safety risks associated with CAR-T/CAR-NK cell therapies. The concept is novel, and the initial in vivo efficacy data are encouraging. However, several significant limitations in experimental design, mechanistic depth, and evidence rigor temper the strength of the conclusions.

Strengths:

(1) Novel conceptual approach.

The adaptation of CAR targeting principles to extracellular vesicles represents a creative and potentially impactful strategy. By displaying CD19 scFv on NK-92-derived vesicles, the authors successfully confer B cell-targeting capability while retaining the cytotoxic effector functions of the parental NK cells. This "cell-free" concept addresses genuine limitations of live cell therapies, including the need for lymphodepletion, risks of cytokine release syndrome, and manufacturing complexity.

(2) Comprehensive in vivo efficacy readouts.

The study evaluates therapeutic effects across multiple clinically relevant endpoints: B cell depletion (flow cytometry), renal function (proteinuria, UPCR), renal histopathology (HE staining with semi-quantitative scoring), systemic inflammation (IgE, IL-17A, IFN-γ), autoantibody production (anti-dsDNA, ANA), and survival. This multi-dimensional characterization strengthens the phenotypic evidence for efficacy.

(3) Appropriate control groups.

The inclusion of non-targeted NK92-Exo as a control allows attribution of the observed effects to CD19-mediated targeting rather than non-specific vesicle-associated activities.

(4) Significant survival benefit.

The improvement in survival from 25% to approximately 80% in V-CD19-Exo-treated mice is substantial and represents arguably the most compelling evidence for therapeutic potential in this model.

Weaknesses:

(1) Mechanism of B-cell reduction remains unclear.

The manuscript reports a dramatic reduction in splenic CD19+CD20+ B cells (from 10.53% to 1.51%) following V-CD19-Exo treatment. However, the authors do not establish whether this results from direct cytotoxicity (e.g., perforin/granzyme-mediated killing, apoptosis induction) or from functional suppression/downregulation of CD19 expression. The authors speculate that the effect is likely mediated by cytotoxic proteins carried by NK-92-derived vesicles, but no data are provided to support this mechanism. Essential experiments would include the detection of apoptosis markers (Annexin V, activated caspase-3/7) in B cells, assessment of perforin/granzyme B content within V-CD19-Exo, or in vitro co-culture assays demonstrating direct B cell killing.

We sincerely thank the reviewer for raising this excellent question. We fully agree that it is an important point that truly needs to be addressed. We are pleased to confirm that we have already begun investigating this and hope to obtain meaningful results in due course.

(2) Small sample sizes.

Most experimental endpoints were assessed with n=5 per group, which is marginal for detecting modest effect sizes and may amplify the influence of individual biological variation. While the survival study had n=10 per group, the main mechanistic and endpoint analyses would benefit from larger cohorts (n=8-10) to increase statistical power and robustness.

We are most grateful to the reviewer for this thoughtful and constructive comment. We completely agree that the sample size in our current analysis is somewhat limited for robust statistical evaluation. We are pleased to report that we have since collected additional data, which we will incorporate into our revised manuscript to strengthen the statistical power. If further data become available, we will gladly update them in subsequent revisions.

(3) No dose-response or dosing optimization studies.

All experiments used a single dose (10⁹ particles per injection) and a fixed schedule (twice weekly for three weeks). The absence of dose-response data leaves unclear whether the observed effects represent maximal efficacy or could be achieved with lower doses, and whether alternative dosing regimens could improve outcomes or reduce potential off-target effects.

We appreciate the reviewer's thoughtful and important question. We completely agree that this needs to be addressed, and we have already started working on it. We will be pleased to update our data in later comments once further results are obtained.

(4) Lack of safety assessment.

The authors emphasize the theoretical safety advantages of extracellular vesicles over cell therapies, but no systematic safety evaluation is presented. Key missing data include: histopathological examination of non-target organs (liver, lung, heart, gastrointestinal tract), assessment of off-target immune activation (T cell responses, cytokine profiles beyond those measured), and evaluation of potential accumulation or toxicity with repeated dosing.

We appreciate the reviewer's careful and important observations. We fully agree that a systematic safety assessment is necessary.We are actively conducting these experiments and will update our manuscript with the findings as soon as possible.

(5) Incomplete characterization of the engineered vesicles beyond targeting.

While the manuscript successfully demonstrates CD19scFv display and vesicle enrichment of exosomal markers, it does not characterize whether V-CD19-Exo retains the full spectrum of NK-92 effector molecules (perforin, granzymes, FasL, TRAIL, cytokines such as IFN-γ) at functional levels. Quantitative or semi-quantitative comparison of cargo between V-CD19-Exo and parental NK-92 cells or non-engineered NK92-Exo would help contextualize the observed in vivo effects.

We thank the reviewer for this valuable comment. We fully agree that further characterization of the engineered vesicles including NK-92 effector molecules and cargo comparison is needed. We are actively working on this and will update the manuscript as soon as the data become available.

(6) Sex as a biological variable is not systematically addressed.

The authors note in the Discussion that the same treatment showed more significant efficacy in male mice compared to females (data not shown), yet all main experiments were conducted exclusively in female mice. Given the strong sex bias in SLE epidemiology (approximately 9:1 female-to-male ratio) and potential differences in immune responses between sexes, this observation warrants systematic investigation rather than a footnote. Presenting the sex-differential data or alternatively, conducting adequately powered sex-stratified analyses would substantially strengthen the manuscript.

We appreciate the reviewer's important comment. We agree that sex is a relevant biological variable, but a systematic analysis is beyond the current scope. We will consider this for future studies and will acknowledge this limitation in the Discussion.

(7) Translational claims are premature.

The manuscript repeatedly emphasizes advantages over cell therapy (low immunogenicity, scalable production, no requirement for lymphodepletion) as if these are established properties of V-CD19-Exo. However, no experiments directly compare V-CD19-Exo to CAR-NK or CAR-T cells in terms of efficacy, immunogenicity, or safety. Similarly, claims of "scalable production" and "high batch-to-batch consistency" are not supported by any manufacturing or quality control data. These statements should be toned down or supported with empirical evidence.

We thank the reviewer for this important observation. We fully agree that our therapeutic claims are premature without direct comparative and manufacturing data. We will revise the manuscript to temper these statements and present them as potential advantages that warrant future investigation.

Reviewer #3 (Public review):

Summary:

This manuscript describes the development of engineered NK-92-derived extracellular vesicles (EVs) displaying CD19scFv for targeted treatment of systemic lupus erythematosus (SLE). Using a CD19scFv-LAMP2B fusion strategy, the authors generated EVs intended to selectively target pathogenic B cells in the MRL/lpr lupus mouse model. The study reports reductions in CD19⁺CD20⁺ B-cell populations, improvements in proteinuria and renal histopathology, decreased inflammatory cytokines and autoantibody levels, reduced splenomegaly, and improved survival outcomes following treatment. The work aims to position engineered EVs as a cell-free alternative to CAR-T/CAR-NK therapies for autoimmune disease treatment. While the concept is interesting and potentially translational, the study currently lacks sufficient methodological rigor, EV purification standards, mechanistic validation, and comprehensive characterization to fully support many of the claims presented.

Strengths:

(1) The study addresses an important unmet clinical need in systemic lupus erythematosus and explores an innovative cell-free therapeutic strategy.

(2) The concept of combining CAR-like targeting approaches with engineered EVs is interesting and potentially translational.

(3) The manuscript includes both in vitro and in vivo experiments, including functional renal assessments, immune profiling, histopathology, and survival studies.

(4) The authors attempt to evaluate multiple disease-associated readouts, including proteinuria, cytokines, autoantibodies, splenomegaly, and survival outcomes, which strengthens the overall biological relevance of the work.

(5) The use of engineered NK92-derived vesicles as a scalable alternative to CAR-NK therapy represents a potentially attractive therapeutic platform.

(6) The in vivo therapeutic observations in the MRL/lpr lupus model are encouraging and warrant further mechanistic investigation.

Weaknesses:

(1) The EV isolation strategy is not sufficiently rigorous for defining the isolated particles as "exosomes" according to current International Society for Extracellular Vesicles/MISEV guidelines. The precipitation-based workflow without density gradient purification or SEC raises major concerns regarding EV purity and identity.

We thank the reviewer for this valuable and timely comment. We fully agree that our precipitation-based isolation does not meet MISEV guidelines for defining particles specifically as 'exosomes.' Since our characterization is based on shape, protein markers, and size, we will replace 'exosome' with 'extracellular vesicles' throughout the manuscript to more accurately reflect our methodology.

(2) No direct validation was provided demonstrating successful surface localization or functional accessibility of CD19scFv on EV membranes.

We thank the reviewer for this valuable point. We agree, and we are happy to confirm that we have obtained data on surface localization and functional accessibility of CD19 scFv, which we will include in the revision.

(3) The characterization of EVs is incomplete and insufficient. Additional positive/negative EV markers, purity metrics, and orthogonal characterization methods are required.

We thank the reviewer for this important point. We fully agree that more comprehensive EV characterization is needed. We are pleased to confirm that we have obtained data on CD19 scFv surface localization and accessibility, which we will include in the revision. We also acknowledge the need for additional markers and purity metrics, and will address this as a limitation in the Discussion.

(4) The absence of density gradient ultracentrifugation is particularly concerning, given the systemic injection of EV preparations into mice, as contaminating soluble factors and non-vesicular particles may contribute to the observed therapeutic effects.

We sincerely thank the reviewer for raising this important technical concern. We fully agree that density gradient ultracentrifugation is a more rigorous method for EV purification and that contaminating soluble factors or non-vesicular particles cannot be completely ruled out in our current preparation. We also acknowledge that even with gradient ultracentrifugation, absolute purity is not guaranteed. Nevertheless, we respectfully note that the therapeutic effect of CD19 scFv from EVs was evident when compared to appropriate controls, suggesting that the observed efficacy is attributable at least in part to the EVs themselves. We will add a clear statement of this limitation in the Discussion and will consider more stringent purification methods in our future studies.

(5) The manuscript lacks adequate mechanistic studies explaining how engineered EVs mediate B-cell depletion or immune modulation.

We thank the reviewer for this important point. We agree that mechanistic studies would be valuable, but we respectfully note that our current paper focuses on establishing a proof-of-concept. We plan to investigate the mechanisms of B-cell reduction and immune modulation in our future work.

(6) The in vitro functional assays are weakly designed, particularly the use of A549 cells for evaluating CD19-targeted vesicle function.

We thank the reviewer for this comment. We wish to clarify that the A549 experiment was intended to confirm that the engineered EVs retain their native function, not to validate CD19 targeting (which will be addressed in point (2). We will revise the manuscript to make this distinction clearer.

(7) Important methodological details are missing, including EV normalization strategies, flow cytometry gating controls, blinding procedures, and randomization approaches.

We thank the reviewer for this important observation. We agree that several methodological details were missing. We will reorganize and expand the Methods section to include EV normalization, flow cytometry gating controls, blinding, and randomization procedures.

(8) Several figures, particularly TEM and western blot images, are of low quality and difficult to interpret.

We thank the reviewer for this comment. We agree that the TEM and Western blot images are of low quality. We will provide improved, higher-resolution images in the revision

(9) The study does not sufficiently exclude the possibility that observed therapeutic effects result from contaminating soluble immune mediators rather than EV-specific activity.

We appreciate this concern. Based on our data, we believe the effects are EV-specific. We will acknowledge this limitation and plan additional controls in future work.

(10) Broader immune profiling is lacking despite the systemic immune complexity of SLE.

We thank the reviewer for this important point. We agree that broader immune profiling would be valuable, especially for clinical translation. However, our current study is designed as a proof-of-concept to establish feasibility. We will acknowledge this limitation in the Discussion and plan to address immune profiling in our future work.

(11) The statistical analysis section includes tests that are not reflected in the Results section, creating concerns regarding data presentation and consistency.

We thank the reviewer for pointing this out. We agree that the statistical tests in the Methods do not match those in the Results. We will revise both sections to ensure consistency throughout.

(12) Overall, while the concept is interesting, the manuscript currently falls short of the experimental rigor expected for high-impact translational EV studies.

We sincerely thank the reviewer for this thoughtful comment. We fully agree that this is a very early-stage translational study, and we acknowledge that considerable work remains before any clinical application can be envisioned. Nevertheless, we respectfully believe that our findings provide a valuable conceptual framework and an initial proof-of-concept that may inform and guide future translational development."