Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public Review):
Summary:
This study analyzed biomarker data from 28 subjects with geographic atrophy (GA) in a Phase I/II clinical trial of PPY988, a subretinal AAV2 complement factor I (CFI) gene therapy, to evaluate pharmacokinetics and pharmacodynamics. Post-treatment, a 2-fold increase in the vitreous humor (VH) FI was observed, correlating with a reduction in FB breakdown product Ba but minimal changes in other complement factors. The aqueous humor (AH) was found to be an unreliable proxy for VH in assessing complement activation. In vitro assays showed that the increase in FI had a minor effect on the complement amplification loop compared to the more potent C3 inhibitor pegcetacoplan. These findings suggest that PPY988 may not provide enough FI protein to effectively modulate complement activation and slow GA progression, highlighting the need for a thorough biomarker review to determine optimal dosing in future studies.
Strengths:
This manuscript provides critical data on the efficacy of gene therapy for the eye, specifically introducing complement FI expression. It presents the results from a halted clinical trial, making sharing this data essential for understanding the outcomes of this gene therapy approach. The findings offer valuable insights and lessons for future gene therapy attempts in similar contexts.
Weaknesses:
No particular weaknesses. The study was carefully performed and limitations are discussed.
I have just some concerns about the methodology used. The authors use the MILLIPLEX assays, which allow for multiplexed detection of complement proteins and they mention extensive validation. How are the measurements with this assay correlating with gold standard methods? Is the specificity and the expected normal ranges preserved with this assay? This also stands for the Olink assay. Some of the proteins are measured by both assay and/or by standard ELISA. How do these measurements correlate?
The authors thank the reviewer for the positive response. Regarding the ELISA assays used to measure the array of complement proteins described, these were extensively validated for the following parameters: specificity, intra-assay and inter-assay precision, accuracy, stability, reference range, and parallelism. All assays were validated in plasma, vitreous and aqueous humour. Due to the limited volume and availability of ocular fluids from individuals in the study, validation in vitreous and aqueous matrices was performed using a pool of several samples from post-mortem donors. At the time this study was initiated, the Millipore Luminex complement panels and the Quidel C3a and Ba EIA were the most sensitive assays and the only commercially available options capable of measuring the proteins of interest in the context of limited vitreous and aqueous humor sample. The concentrations measured were observed at similar ranges as those published in the literature using assays in distinct patient populations e.g. in (Mandava et al, Invest Ophthalmol Vis Sci, 2020).
Measurements from vitreous and aqueous from subject samples were deemed reportable if they were within the quantifiable ranges defined for these sample types during the validation (coefficient of variation of 20%, or 30% when results were below the lower limit of quantification but above limit of detection). Notably, given the limited amount of biomarker data due to small sample size, we share results from outlier biomarker measurements, to illustrate the heterogeneity in sample quality. We further publish plasma sample biomarker results in supplemental table 5 wherein complement protein concentrations can be observed and compared to normal ranges in the literature.
Adding confidence to the robustness of our assays was the observation that some of the complement proteins quantified by standard assay (e.g. plate and bead-based ELISAs) were also measured by the OLINK assay, and there was a general trend observed for positive correlation between results from both assays for FI levels post-treatment. However, we did not provide detailed correlative statistical analyses for further complement proteins as OLINK findings were deemed highly exploratory and hypothesis generating, and because the OLINK assay produced normalised results which are challenging to directly compare to ELISA results that were absolute.
Reviewer #2 (Public Review):
Summary:
The results presented demonstrate that AAV2-CFI gene therapy delivers long-term and marginally higher FI protein in vitreous humor that results in a concomitant reduction in the FB activation product Ba. However, the lack of clinical efficacy in the phase I/II study, possibly due to lower in vitro potency when compared to currently approved pegcetacoplan, raises important considerations for the utility of this therapeutic approach. Despite the early termination of the PPY988 clinical development program, the study achieved significant milestones, including the implementation of subretinal gene therapy delivery in older adults, complement biomarker comparison between serial vitreous humor and aqueous humor samples and vitreous humor proteomic assessment via Olink.
Strengths:
Long-term augmentation of FI protein in vitreous humor over 96 weeks and reduction of FB breakdown product Ba in vitreous humor suggests modulation of the complement system. Developed a novel in vitro assay suggesting FI's ability to reduce C3 convertase activity is weaker than pegcetacoplan and FH and may suggest a higher dose of FI will be required for clinical efficacy. Warn of the poor correlation between vitreous humor and aqueous humor biomarkers and suggest aqueous humor may not be a reliable proxy for vitreous humor with regard to complement activation/inhibition studies.
Weaknesses:
The vitrectomy required for the subretinal route of administration causes a long-term loss of total protein and may influence the interpretation of complement biomarker results even with normalization. The modified in vitro assay of complement activation suggests a several hundred-fold increase in FI protein is required to significantly affect C3a levels. Interestingly, the in vitro assay demonstrates 100% inhibition of C3a with pegcetacoplan and FH therapeutics, but only a 50% reduction with FI even at the highest concentrations tested. This observation suggests FI may not be rate-limiting for negative complement regulation under the in vitro conditions tested and potentially in the eye. It is unclear if pharmacokinetic and pharmacodynamic properties in aqueous humor and vitreous humor compartments are reliable predictors of FI level/activity after subretinal delivery AAV2-CFI gene therapy.
The authors thank the reviewer for the positive response and we agree that a limitation of the biomarker strategy for ocular gene therapy delivered to the retinal tissues is inferring PK/PD from vitreous and aqueous samples, which are the fluid sample compartments accessible from subjects available to measure molecular treatment response. We agree that these compartments may not accurately represent sub-retinal and tissue level complement turnover. In the discussion, line 508, we state: ‘Overall, the data suggests that fully functional FI is being secreted into the VH, but the regulatory effects on the level of Ba may be representative of convertase formation in the VH and not the macula retina/RPE nor the choroid. To validate this hypothesis, one approach would be to conduct vitreal sampling using an effective drug targeting C3 for GA in a larger cohort’.
However, the observation of elevation of FI in VH (and AH) post treatment, and changes in levels of downstream complement proteins that align with prior knowledge of control of alternative pathway activation, is compelling evidence that these measurements reflect modest but direct consequences of an FI-gene therapy that was delivered to the subretinal space. We add to the discussion, line 479: ‘the findings of elevated FI in the VH after sub-retinally delivered CFI gene therapy and changes in complement pathway proteins post-treatment build confidence that VH matrix is at least partially reflecting the complement system at the retinal layers and treatment site, and is a valid biomarker for PK/PD insights in response to treatment.’
Furthermore, the observation of moderately raised FI levels in modelled VH post treatment being insufficient to control CS activation in vitro accords with the lack of clinical response observed at phase II. We note that measuring FI and complement biomarkers in retinal tissues from treated eyes at post-mortem would be one way to explore the PK/PD effects from AAV2-FI gene therapy.
Reviewer #3 (Public Review):
Summary:
The manuscript by Hallam et al describes the analysis of various biomarkers in patients undergoing complement factor I supplementation treatment (PPY988 gene therapy) as part of the FOCUS Phase I/II clinical trial. The authors used validated methods (multiplexed assays and OLINK proteomics) for measuring multiple soluble complement proteins in the aqueous humour (AH) and vitreous humour (VH) of 28 patients over a series of time points, up to and including 96 weeks. Based on biomarker comparisons, the levels of FI synthesised by PPY988 were believed to be insufficient to achieve the desired level of complement inhibition. Subsequent comparative experiments showed that PPY988-delivered FI was much less efficacious than Pegceptacoplan (FDA-approved complement inhibitor under the name SYFORVE) when tested in an artificial VH matrix.
Strengths:
The manuscript is well written with data clearly presented and appropriate statistics used for the analysis itself. It's great to see data from real clinical samples that can help support future studies and therapeutic design. The identification that complement biomarker levels present in the AH do not represent the levels found in the VH is an important finding for the field, given the number of complement-targeting therapies in development and the desperate need for good biomarkers for target engagement. This study also provides a wealth of baseline complement protein measurements in both human AH and VH (and companion measurements in plasma) that will prove useful for future studies.
Weaknesses:
Perhaps the conclusions drawn regarding the lack of observed efficacy are not fully justified. The authors focus on the hypothesis that not enough FI was synthesised in these patients receiving the PPY988 gene therapy, suggesting a delivery/transduction/expression issue. But beyond rare CFI genetic variants, most genetic associations with AMD imply that it is a FI-cofactor disease. A hypothesis supported by the authors' own experiments when they supplement their artificial VH matrix with FH and achieve a significantly greater breakdown of C3b than achieved with PPY988 treatment alone. Justification around doubling FI levels driving complement turnover refers to studies conducted in blood, which has an entirely different complement protein profile than VH. In Supplemental Table 5 we see there is approx. 10-fold more FH than FI (533ug/ml vs 50ug/ml respectively) so increasing FI levels will have a direct effect. Yet in Supplemental Table 3 we see there is more FI than FH in VH (608ng/ml vs 466ng/ml respectively). Therefore, adding more FI without more co-factors would have a very limited effect. Surely this demonstrates that the study was delivering the wrong payload, i.e. FI, which hit a natural ceiling of endogenous co-factors within the eye?
See response to reviewer 3’s review after reviewer 3 recommendations section below.
Recommendations for the authors:
Reviewer #2 (Recommendations For The Authors):
The authors present strong evidence using validated complement biomarker assays and comprehensive proteomic profiling that support their findings. The presentation of complement biomarker data in vitreous humor and aqueous humor after FI augmentation is presented in a clear and concise format. The direct comparison of complement biomarkers in vitreous humor and aqueous humor from the same patients and demonstrating similarities and differences is important for the nascent complement gene therapy field. Developing a novel in vitro complement model and comparing pegcetacoplan, FH, and FI inhibitors provides the field with a valuable assay to benchmark other complement therapeutics. As currently designed, the in vitro assay supports why FI augmentation did not contribute to clinical success. It also suggests that non-physiological concentrations of FI protein (over 100 µg/mL) maximally inhibit C3a signal by ~50%, whereas both pegcetacoplan and FH reduce the signal by 100%. Does this suggest that CFI is not an appropriate therapeutic target to control complement overactivation in the eye?
We agree with the reviewer that the new data from the novel in vitro assay coupled with the clinical findings from the phase II gene therapy trial does now suggest FI is less attractive as a therapeutic target for controlling complement activation in the retinal tissues of subjects with Geographic Atrophy.
Reviewer #3 (Recommendations For The Authors):
I think the authors have done a great job collecting and analysing these clinical samples and elucidating the baseline complement protein profile in both the AH and VH. I only have minimal suggested changes.
Perhaps a more direct discussion around the limitations of adding more FI into environments where there is no excess of FI-cofactors present? And a discussion around the limitations of VH (and VA for that matter) biomarker sampling for a disease that primarily affects the neurosensory retina and outer blood/retinal barrier: perhaps the landscape of complement proteins is different yet again (although, admittedly, impossible to sample in a patient)? Finally, would it not have been better to perform complement activation experiments using the VH of treated patients directly rather than creating an artificial VH matrix (there may, or may not, be a couple of things in human VH that directly affect complement turnover...)?
We thank the reviewer for the supportive comments. This study is the first to describe FI and FH levels and respective ratios in vitreous humour (plus aqueous and plasma) from GA subjects, before and after sub-retinal gene therapy. It is compelling to observe that in the VH the levels of FI are greater than FH, the primary fluid phase co-factor for FI enzymatic activity. This new information does indeed argue against further FI supplementation (using gene therapy) being of added benefit to controlling the complement system in the broader population in individuals with Geographic Atrophy. We note that at the start of the clinical development of GT005/PPY988 AAV2-FI gene therapy, there was limited information on FI and FH levels in AMD in ocular fluids to inform the pharmacodynamics of complement activation. Now, by running the FOCUS phase I clinical trial and measuring the complement biomarker data using validated assays we have added to our understanding on the levels and ratio of FI to FH and other complement proteins in a larger number of GA subjects’ ocular samples. We report the levels of complement proteins measured in ocular and systemic samples, to show the ranges and also the differences in ratios between the different matrices.
Regarding the statement that FI supplementation could likely be ineffective due to limited FH cofactor; FH is not the only co-factor that FI may partner with at cell surfaces to become enzymatically active (others include MCP (CD46) and CR1 (CD35), although the latter is known to be of limited expression in the eye), as such, it is certainly true that other proteins may be present in the tissue altering the kinetics of FI’s activity after sub-retinal gene-therapy. In addition, the ratio between FI and FH detected in the VH may not be the same as in retinal tissue. As such, we agree that drawing insights from biomarkers in the VH may not fully reflect the disease processes and treatment response at the retinal cell layers, but it is the closest fluid sample available to sample tissue released soluble proteins. We acknowledge that VH biomarkers will not fully capture retinal disease processes and treatment responses, but due to their proximity, will reflect retina-released soluble proteins. The findings of elevated FI in the VH after sub-retinally delivered CFI gene therapy and changes in complement pathway proteins post-treatment build confidence that VH matrix is at least partially reflecting the complement system at the retinal layers and treatment site, and is a valid biomarker for PK/PD insights in response to treatment. We agree modelling different inhibitor effects on complement activation directly using subject’s vitreous would be informative, but this was not possible due to the limitations of very small sample volume.
We add several sentences to the discussion regarding the points above. Line 473: ‘Notably, that FI does not reduce C3a breakdown to baseline even at supermolecular concentrations suggests cofactor limitation that might be more pronounced in VH given FH is not in excess of FI as is the case in blood 27. Moreover, there are additional cell-bound cofactors for FI that may be present in retinal tissue that are not present in the VH and could further alter the kinetics of the assay, such as MCP (CD46) albeit with disease related changes observed 37. However, the findings of elevated FI in the VH after sub-retinally delivered CFI gene therapy and changes in complement pathway proteins post-treatment build confidence that VH matrix is at least partially reflecting the complement system at the retinal layers and treatment site, and is a valid biomarker for PK/PD insights in response to treatment.’
Minor comments:
Line 237: Missing parenthesis at the end of the sentence
Manuscript updated.
Line 435: Missing secondary parenthesis after .....Figure 3A)......
Manuscript updated.
Line 536: I don't think suggesting the addition of FHR proteins into the neurosensory retina/VH is such a good idea
The reference to FHRs has been clarified in the manuscript, line 558. The authors note that FHR dimerization domains have been engineered to dimerize Factor H constructs increasing half-life and potency for drugs currently in development.
