Molecular Survival Strategies Against Kidney Filtration: Implications for Therapeutic Protein Engineering

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Very interesting review article. The paper was propable preparing by young, not medical scientists , but is useful also for doctors . It  is modern , complete view

 

Author Response

Reviewer 1

Comment 1:Very interesting review article. The paper was propable preparing by young, not medical scientists, but is useful also for doctors. It is modern, complete view.

Response: We thank the reviewer for their analysis of the paper. We agree that it may be valuable to both the academic and medical communities.

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript provides a comprehensive and well-structured review of endogenous and artificial strategies used to overcome renal clearance of therapeutic proteins. The authors successfully integrate renal physiology, protein biophysics, and translational protein engineering, making the review timely and relevant for researchers involved in biologics development, pharmacokinetics, and protein design.

The article is well written, logically organized, supported by an extensive and up-to-date list of references, with informative figures and tables that help synthesize complex concepts. Overall, this is a strong review that would be suitable for publication after minor to moderate revisions aimed at improving balance, clarity, and critical depth.

Please accept the following comments

Comment 1

While the emphasis on glomerular filtration is appropriate and clearly stated, the manuscript sometimes risks attributing systemic clearance too strongly to renal mechanisms alone, without considering integrating other clearance pathways. Although hepatic uptake, target-mediated drug disposition and proteolytic degradation are mentioned, they are not uniformly incorporating throughout all sections, particularly when larger biologic formats are taking into consideration.

Suggestion:

Add a brief unifying paragraph to the introduction section, clarifying how renal filtration interacts with other clearance mechanisms, particularly with regard to Fc-binding and albumin-based strategies, where non-renal pathways predominate.

 

 

Comment 2:

Immunogenicity is discussed in multiple sections throughout the manuscript and is mostly qualitative. Due to its clinical relevance, please do include a short paragraph on immunogenic risk profiles of e.g PEGylation vs. Fc-fusions and clinical consequences of anti-drug antibodies (i.e loss of efficacy vs. hypersensitivity)

Author Response

Reviewer: 2

Summary:

This manuscript provides a comprehensive and well-structured review of endogenous and artificial strategies used to overcome renal clearance of therapeutic proteins. The authors successfully integrate renal physiology, protein biophysics, and translational protein engineering, making the review timely and relevant for researchers involved in biologics development, pharmacokinetics, and protein design.

The article is well written, logically organized, supported by an extensive and up-to-date list of references, with informative figures and tables that help synthesize complex concepts. Overall, this is a strong review that would be suitable for publication after minor to moderate revisions aimed at improving balance, clarity, and critical depth.

Response: We thank the reviewer for their comment regarding the aim and application of the review and are glad to know that they found it to be valuable. We acknowledge that there are revisions to be made that will further deepen the impact of the paper.

Comment 1: While the emphasis on glomerular filtration is appropriate and clearly stated, the manuscript sometimes risks attributing systemic clearance too strongly to renal mechanisms alone, without considering integrating other clearance pathways. Although hepatic uptake, target-mediated drug disposition and proteolytic degradation are mentioned, they are not uniformly incorporating throughout all sections, particularly when larger biologic formats are taking into consideration.

Suggestion 1: Add a brief unifying paragraph to the introduction section, clarifying how renal filtration interacts with other clearance mechanisms, particularly with regard to Fc-binding and albumin-based strategies, where non-renal pathways predominate.

Response: We appreciate the suggestion given by the reviewer and agree that additional clarification regarding other clearance pathways would be beneficial. Accordingly, we have added the suggested paragraph to the introduction (lines 55-): “Therapeutics that are too large to be filtered renally are cleared via other pathways. For instance, those with exposed regions or conformational flexibility may be subject to proteolytic degradation by circulating or tissue-associated proteases. Molecules may also be internalized via receptor-mediated endocytosis and directed to lysosomes for degradation. Proteins that bear hydrophobic patches or non-native glycan structures are frequently cleared through hepatic uptake. In addition, therapeutics that elicit immunogenic responses are rapidly cleared by anti-drug antibodies that form a complex with the therapeutic, promoting Fc receptor-mediated uptake and phagocytosis. While these pathways are important to consider in designing biologics, renal filtration is the primary concern of many modern protein and peptide therapeutics due to their small size.”

 

Suggestion 2: Immunogenicity is discussed in multiple sections throughout the manuscript and is mostly qualitative. Due to its clinical relevance, please do include a short paragraph on immunogenic risk profiles of e.g PEGylation vs. Fc-fusions and clinical consequences of anti-drug antibodies (i.e loss of efficacy vs. hypersensitivity)

Response: We agree that another subsection comparing the immunogenic risks of each strategy would be advantageous. We have added a subsection (starting on line 535) to Section 3 with this suggestion in mind: “A common challenge in extending the circulating half-life of therapeutics is the risk of immunogenic responses, which generally increases with the degree of structural deviation from endogenous molecules. Although PEGylation was initially considered non-immunogenic, accumulating evidence demonstrates the formation of anti-PEG antibodies that can accelerate clearance and reduce therapeutic efficacy, particularly upon repeated dosing, where antibody titers may increase over time. Consequently, patient screening and immunogenicity monitoring may be important considerations for PEGylated biologics, especially in chronic treatment settings. In contrast, albumin-based strategies and Fc fusion proteins typically exhibit low immunogenic risk, as both human serum albumin and the IgG Fc region are endogenous. Anti-drug antibody incidence is generally low, and antibodies are most often non-neutralizing with minimal impact on pharmacokinetics or efficacy, although albumin-binding domains derived from bacterial proteins represent a notable exception due to their potential to elicit immune responses. The immunogenicity of glycoengineered biologics is highly dependent on glycan composition, with human-like glycans being well tolerated, whereas non-human sugars such as α-galactose or N-glycolylneuraminic acid can provoke strong anti-drug antibody or hypersensitivity reactions. Finally, unstructured polypeptide extensions are intentionally designed to minimize immunogenicity by avoiding sequence motifs associated with immune recognition and have not been observed to induce significant anti-drug antibody formation in preclinical and early clinical studies.”

 

 

 

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript demonstrates high scientific value and adequate documentation.

The conceptual organization is logical, and the rigor is high. Molecular mechanisms are correlated with clinical implications. Tables and figures are clear and of high quality.

As a suggestion, although the manuscript is very comprehensive, the introduction could better explain the formulation of the elements that distinguish this review from other similar works in the specialized literature. More specifically, the biology of renal filtration, along with its engineering implications and computational models, should be more clearly defined.

Regarding the discussion on charge selectivity, the controversy regarding the physiological relevance of electrostatic selectivity is correctly emphasized. However, a conclusion that synthesizes the experimental data in accordance with in vivo observations could be beneficial.

In the section on de novo design, it is necessary to complete it with a brief discussion of the current experimental limitations in accordance with clinical applicability.

It would also be useful to include references directly in tables or footnotes.

The paper may be accepted after minor revisions.

Comments on the Quality of English Language

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Author Response

Reviewer: 3

Comment 1: The manuscript demonstrates high scientific value and adequate documentation.

Response: We thank the reviewer for their analysis of this paper and are pleased to know that they find it scientifically valuable and adequately documented.

Comment 2: The conceptual organization is logical, and the rigor is high. Molecular mechanisms are correlated with clinical implications. Tables and figures are clear and of high quality.

Response: We thank the reviewer for their comment on the rigor and organization of this review. We are glad that the correlations and implications we drew were clear and that the tables and figures accomplished their respective purposes.

Suggestion 1: As a suggestion, although the manuscript is very comprehensive, the introduction could better explain the formulation of the elements that distinguish this review from other similar works in the specialized literature. More specifically, the biology of renal filtration, along with its engineering implications and computational models, should be more clearly defined.

Response: We agree with the reviewer that further clarifying and defining the distinguishing features of this review would be beneficial to readers. Accordingly, we have added a portion of text to the end of the introduction (lines 87-92): “While previous reviews have described strategies for extending the half-life of biologics, this review uniquely grounds these approaches in the anatomy and physiology of the renal filtration barrier to explain why they are effective. It further compares the strategies with endogenous survival mechanisms and highlights how emerging computational tools may be leveraged to further extend the circulatory half-life of therapeutics in vivo.”

Suggestion 2: Regarding the discussion on charge selectivity, the controversy regarding the physiological relevance of electrostatic selectivity is correctly emphasized. However, a conclusion that synthesizes the experimental data in accordance with in vivo observations could be beneficial.

Response: We thank the reviewer for their appreciation of our explanation of the electrostatic selectivity controversy and agree that a practical conclusion based on in vivo observations would be of value to this paper. We have added a conclusion at the end of section 2.3.1 (lines 232-239) to accomplish this: “Overall, the evidence supports electrostatic selectivity as a real but secondary component of glomerular permeability. While tracer studies clearly demonstrate preferential filtration of cationic macromolecules, in vivo models show that changes in barrier charge result in only modest proteinuria when size selectivity is preserved. These findings suggest that charge primarily fine-tunes filtration by enhancing retention of anionic proteins and accelerating clearance of cationic species, rather than serving as the dominant physiological barrier. This synthesis reconciles experimental observations with in vivo behavior and explains the continued relevance of charge in therapeutic protein design.”

Suggestion 3: In the section on de novo design, it is necessary to complete it with a brief discussion of the current experimental limitations in accordance with clinical applicability.

Response: We thank the reviewer for drawing our attention to the need for a more thorough discussion of the limitations of de novo protein design. We have added a paragraph discussing these limitations to the end of section 4.2 (lines 599-609): “Despite these advantages, the clinical translation of de novo designed therapeutics remains in its nascent stages. A primary challenge lies in immunogenicity; while computational methods can screen for known epitopes, novel sequences possess the risk of eliciting anti-drug antibodies upon repeated dosing. Furthermore, it can be difficult to ensure solubility and prevent aggregation at high clinical concentrations, even after computational optimization. A common critique of de novo designed scaffolds is the stability-activity tradeoff. Backbones designed de novo tend to exhibit impressive stability. However, this structural rigidity can impede the dynamics needed for efficient catalysis. While challenges such as immunogenicity, solubility, and the stability-activity tradeoff remain, de novo design offers versatile strategies that will be explored to highlight its substantial potential for clinical application.”

 

Suggestion 4: It would also be useful to include references directly in tables or footnotes.

Response: We agree that references should be contained within the tables and figures. References are found both in the tables and figure descriptions (see lines 44, 115, and Table 1).