Hello, ladies and gentlemen. Thank you for standing by, and welcome to the 4D Molecular Therapeutics Webcast Presentation of Interim Data from the 4D-710 Phase I/II AEROW Clinical Trial in cystic fibrosis. As a reminder, today's call is being recorded. With that, I will hand over the call to Uneek Mehra, Chief Financial and Business Officer, who will make introductory comments.
Thank you, operator, and welcome to our webcast. We issued a press release describing the 4D-710 AEROW interim results earlier today, and the recording of this webcast will be accessible in the Investors section of the 4DMT website after the completion of this call. With me today are Dr. David Kirn, our Co-founder and Chief Executive Officer, who will provide introductory remarks. Dr. Alan Cohen, SVP Therapeutic Area Head of Pulmonology, who will review the program summary and next steps; and Dr. Jennifer Taylor-Cousar, Professor and Co-director, Adult Cystic Fibrosis Program and Director, CFF Therapeutics Development Center, National Jewish Health, who will review the clinical data. She will also present the interim data later today at the 2024 European Cystic Fibrosis Conference in Glasgow, U.K. We remind you here that we may be making forward-looking statements. For further details, you can visit our website.
With that, I would like to turn the call over to our CEO, David Kirn. David?
Thank you, Uneek, and thank you everyone for joining us today. We're pleased to share positive new data from the phase I dose exploration stage of the 4D-710 AEROW clinical trial that supports 4D-710 's potential as a durable genetic medicine for cystic fibrosis lung disease. As a reminder, our wholly owned pulmonology franchise is powered by our aerosolized next-generation A101 vector, invented at 4DMT. In the phase I stage of the AEROW trial, we evaluated 4 dose levels of a single aerosolized administration, ranging from 2.5 × 10^14 to 2 × 10^15 VGs in 10 participants. Here are the 4 key takeaways.
First, lung function, as measured by percent predicted forced expiratory volume in one second, or ppFEV1, improved by 5 and 6 percentage points at 12 months post-dosing in two of the three participants, with greater than 6 months of follow-up and mild to moderate lung function impairment at baseline. Improvements of 5%-6% in ppFEV1 are believed to be in a clinically meaningful range. This population is typically the population studied in CFTR modulators and is the target population for a phase III trial. Second, 4D-710 was well-tolerated and safe in all six participants at doses up to 1 × 10^15 VGs. Third, lung biopsy results demonstrate dose-dependent 4D-710 -mediated transient expression with widespread CFTR protein expression in all participants. To our knowledge, reproducible and widespread CFTR transient expression has never before been reported in humans.
This landmark success demonstrates the power of the 4DMT Therapeutic Vector Evolution platform that leverages the Nobel Prize-winning technology of directed evolution. Finally, widespread transient expression, improved FEV1, and quality of life, plus safety, was shown in participants with pre-existing AAV immunity that cross-reacted with our A101 vector. This data from humans, combined with redosing feasibility data from animal models, strongly supports our belief that redosing with aerosol administration will be biologically active and well-tolerated. A101 was evolved at 4DMT to resist neutralization by pre-existing antibodies in humans. With respect to next steps, based on all available data to date, we are clear to begin enrollment in the phase II dose expansion stage in the second half of 2024, and we plan to enroll up to nine total participants. We plan to begin enrollment with the 1E15 VG dose.
We are also continuing to evaluate 5E14 VGs as a potential additional phase II dose to consider in parallel, and we plan to complete evaluation of this dose in the phase I, with the enrollment and follow-up of the third and final participant in the 5E14 VG cohort. Finally, we plan to provide the next interim data update in mid-2025 after completing enrollment and collecting sufficient follow-up in participants enrolled in phase II. The aerosolized A101 lung vector is one of three novel, highly targeted next-generation AAV vectors invented at 4DMT, with proof-of-concept data from clinical trials. A101 is an important value driver for the company as we advance our pulmonology portfolio, including 4D-710 in cystic fibrosis lung disease and 4D-725 in α-1-AT lung disease. 4DMT owns all worldwide rights to A101-based products.
I will now hand the presentation over to Alan to talk more about CF. Alan?
Thanks, David. To review, CF is caused by genetic variants that result in a dysfunctional cystic fibrosis transmembrane conductance regulator, or CFTR protein. Without properly functioning CFTR, cells are unable to effectively move chloride from inside the cell to the cell surface. This results in thick, tenacious mucus with unremitting airway inflammation, frequent lower airway bacterial infections, and destructive airway events with bronchiectasis, culminating in progressive respiratory failure and, sadly, early death. In the pre-modulator era, this resulted in an annual lung function decline of approximately 1%-2% per year, as measured by percent predicted FEV1. Additionally, the median life expectancy of people with CF in this pre-modulator era was approximately 40 years, reflecting the reality of many modulator-ineligible individuals today. CF affects over 100,000 individuals worldwide and approximately 40,000 in the United States.
People with CF currently receive intensive, time-consuming daily airway clearance therapy, mucolytics, inhaled antibiotics, and bronchodilators. Additionally, for many people with CF, twice-daily oral CFTR modulators can partially restore decreased or absent CFTR function, but these therapies are only available for those who have eligible genetic variants. Modulator therapies have been rapidly adopted and have generated approximately $10 billion in global sales in 2023 alone, and the market continues to grow. We leveraged our therapeutic vector evolution platform to invent A101, a next-generation lung delivery vector. Prior AAV gene therapy approaches for targeted lung delivery have failed, and none of these approaches have achieved meaningful gene expression in CF airways. These approaches were hampered by a variety of limitations of conventional vectors, including poor mucus penetration, inefficient airway cell transduction, suboptimal tissue tropism, and susceptibility to clearance by antibodies.
At 4DMT, we invented A101 to overcome these limitations. It has favorable characteristics, including highly efficient mucus penetration and transgene expression, the ability to transduce multiple airway cell types, including basal or basement membrane cells, high specificity for the lung, and resistance to pre-existing human AAV immunity. On the right panel, as shown in our genetic medicine design schematics, our lung-targeted therapies are comprised of the A101 vector carrying a therapeutic payload for each site-specific genetic disease. We then deliver the genetic medicine using the FDA-approved AeroEclipse II breath-actuated nebulizer. This device produces stable and reproducible droplets in the respirable range, enabling even distribution throughout the large and small airways, including the alveoli. Our initial CF target population, using 4D-710 as a monotherapy, are those people with CF with the highest unmet medical need.
This represents an estimated 15% of all people with CF, of which approximately 10% are not eligible for CFTR modulators and approximately 5% are intolerant to their side effects. In addition, it's estimated that approximately 20% of people who take modulators have less than a 5% improvement in percent-predicted FEV1, and therefore could benefit from additional CFTR function. We believe this population has additional unmet medical need and represents a, a potential future for 4D-710 study population. Lastly, the remaining balance of modulator-treated people with CF that have a demonstrable response could still potentially benefit from 4D-710, given the potential durable nature of a genetic medicine compared to a daily oral therapy.
Ultimately, with 4D-710, we believe we have the opportunity to potentially treat all people with CF, regardless of their CFTR genetic variant, both as a standalone therapy or in combination with current standard of care modulators. I'd like to now hand the presentation over to Dr. Jennifer Taylor-Cousar, lead principal investigator for the trial, to present the study design and early data from AEROW. Thanks for being with us and dialing in all the way from the ECFS meeting in Glasgow, U.K. Dr. Taylor-Cousar?
Thanks, Dr. Cohen. On this slide, we show the design of the phase I/II AEROW clinical trial, evaluating 4D-710 as a single aerosolized dose in adults with CF lung disease who are either ineligible for or do not tolerate CFTR modulator therapy. The goal of the phase I dose exploration stage of the study is to conduct dose ranging to characterize safety, tolerability, biomarkers, and clinical activity with a single administration of 4D-710. Analyses of all study data were to be used to select a dose for the phase II dose expansion stage. We have cleared 1 × 10 to the 15th vector genomes as a phase II dose and plan to begin enrollment in the second half of 2024.
In parallel, we are continuing to evaluate the 5 × 10^14 vector genome dose by completing enrollment and follow-up for a third participant in this dose cohort. Phase II is designed to affirm safety and tolerability of 4D-710 in participants with mild to moderate lung impairment, and to assess the magnitude of clinical activity we might expect to observe in a fully blinded and controlled phase III development program. We expect to enroll up to 9 total evaluable participants, all with baseline percent predicted FEV1 from approximately 50%-90%, potentially allowing us to observe a more pronounced benefit from treatment with 4D-710. In the study diagram below, you can see 4D-710 aerosol administration on day 1.
Bronchoscopy is performed at week 4 to 8 to collect multiple lung tissue samples for assessment of transgene delivery and expression. Transient mild immunosuppression is given as a 40-milligram prednisone treatment, tapered over 4 weeks, starting on day -1. This table shows key baseline characteristics of the 10 participants enrolled across 4 dose cohorts. Ages range from 20 to 69 years old. Three of the 10 participants were intolerant to CFTR modulators, and the remaining 7 participants had variants not amenable to treatment with CFTR modulators. Of note, these participants had a broad range of lung function impairment at baseline, measured by % predicted FEV1. 5 participants had mild to moderate impairment, defined as 40%-80% at baseline.
Lastly, 3 participants had baseline antibodies that cross-reacted with the A101 capsid, and 3 participants had A101 cross-reactive T cells detected for a total of 5 of 10 participants who had preexisting immunity to A101. This slide highlights the safety and tolerability of 4D-710 at the 2 × 10^15 vector genome dose, our highest studied dose level. We have studied 3 lower dosing groups, which will be the focus of future participant enrollments moving forward. A non-serious treatment related adverse event of pneumonitis and percent predicted FEV1 decline was observed in 1 participant treated at this highest dose, 2 × 10^15 vector genomes. The pneumonitis and a diminishment in airflow, as measured by changes in percent predicted FEV1, have both fully resolved.
As previously reported in one participant, a serious adverse event of pneumonitis not otherwise specified has fully resolved, and importantly, this participant's percent predicted FEV1 has improved by 6 percentage points at last assessment at month 12 compared to baseline. As previously reported, analyses of the tissue samples from lung biopsies obtained at weeks 4-8 showed no evidence of inflammation or toxicity. In terms of CFTR protein expression, we observed what we believe to be significant overexpression of this critically important airway protein, measured at approximately 400% higher in the epithelium compared to normal, in other words, non-CF lung samples. There was no increase compared to the second highest dose level of 1 × 10^15 vector genomes. We also observed widespread expression in the interstitium, which was not part of our target expression profile and was not observed in normal lung controls.
Based on these observations, the 2 x 10^15 vector genome dose level will not be further evaluated, and we define 1 x 10^15 vector genomes as the highest dose for further consideration. This slide illustrates the safety data following dosing of 4D-710 in six participants, in the other three lower dose level cohorts, ranging from 1-25 months of follow-up to date. Green boxes illustrate a lack of any reported 4D-710-related adverse events during that month, showing that 4D-710 administered at doses less than or equal to 1 x 10^15 vector genomes have been well tolerated to date. In the 1 x 10^15 vector genome dose cohort, no 4D-710-related adverse events were reported in three of three participants over 22-25 months of follow-up.
In early follow-up of lower dose cohorts ranging from 1 to 7 months, we also saw no 4D-710-related adverse events, except for only one grade 1 adverse event at the 5 × 10^{14} vector genome dose level of increased sputum at month 1. In these three cohorts, after nebulized administration, we saw no dose-limiting toxicities, no 4D-710-related serious adverse events, and no clinically significant 4D-710-related adverse events. Lastly, there was no inflammation or toxicity observed in the tissue samples from lung biopsies. This slide shows the post-dosing bronchoscopy sampling plan scheduled for week 4 to 8. Multiple lung tissue samples are collected for assessment of 4D-710-mediated transgene delivery and expression. The table on the left summarizes the number and location of lung samples collected by biopsies and brushings of both lungs at multiple regions.
The diagram on the right shows the locations of all 4 protocol-defined biopsies in green. These biopsies were taken at the entry to the upper and middle lobes on the right, and at the entry to the upper lobe and lingula on the left. In pink, lung brushing locations are shown in the lower lobes bilaterally. Biopsies were obtained for PCR, specifically for transgene DNA. In addition, both types of samples were obtained to measure transgene RNA expression using in situ hybridization, or ISH, and to measure CFTR protein with immunohistochemistry, or IHC. On this slide, we show that assessment of transgene expression in tissue samples from endobronchial biopsies demonstrated what appears to be linear dose-dependent CFTR transgene mRNA expression in bronchial epithelial cells, measured by mean % of cells positive for expression.
This mean ranged from 14% at the 2.5 x 10^14 vector genome dose, to 53% at the 2 x 10^15 vector genome dose level. In contrast, no CFTR transgene RNA was detected in commercially acquired control samples from CF and non-CF lungs. As one would expect, commercial non-CF samples were positive for endogenous CFTR mRNA expression. Based on this data to date, we believe we have demonstrated a dose response of RNA expression for 4D-710. This slide summarizes our methods for quantifying CFTR protein expression. Quantitation of staining was performed using Visiopharm machine learning-assisted image analysis, which is reliable clinical-grade software. We were able to determine the percent positive cells in H-score that were automatically calculated by the software algorithm. The H-score, with a range of 0-300, is a measure of staining intensity and distribution.
Higher scores indicate increased signal intensity and distribution. Based on preclinical data and the half-life of these molecules, we predicted that IHC for protein would be more sensitive than ISH for RNA. Assessment of CFTR protein expression by IHC showed robust and widespread expression in bronchial epithelial cells at all tested doses. On the left, positive staining was observed in 94%-100% of epithelial cells, and samples collected from study participants, compared to 44% and 18% in commercially acquired non-CF and CF lung samples, respectively. The numbers shown for each group represent the number of biopsy samples. On the right, we show the CFTR staining intensity score, which shows that CFTR protein expression is higher in the epithelium compared to normal, in other words, non-CF lung samples.
In conclusion, we see robust CFTR protein expression following 4D-710 administration in all participants, and this result gives us confidence that we are achieving protein levels that we believe should translate into clinical activity. On this slide, we once again show that CFTR protein expression was observed in 100% of endobronchial biopsy samples in each dose group. Shown here are representative images of tissue samples for each participant. The top row shows images of samples obtained from the left secondary carina, and the bottom shows samples obtained from the right middle lobe. Representative images from commercially acquired non-CF and CF control lung samples are also shown at the bottom of the slide. Widespread staining of bronchial epithelial cells can be seen across all samples.
At the highest tested dose of 2 × 10^15 vector genomes, positive staining was also seen in the interstitium, which is not part of our target expression profile. As previously discussed, this dose level of 2 × 10^15 vector genomes has been discontinued. Here are additional images demonstrating the cell types expressing CFTR. We see CFTR staining in all key cell types, including basal, secretory, and columnar ciliated cells. Importantly, expression in basal cells, which are long-lived stem cells of the lung, will be critical for long-term durability. On the right, you can see the expression is appropriately localized to the apical membrane, which is a key interface between lung epithelial cells and the lung airway lumen.
This slide shows the results of our A101 immunogenicity analyses, which is important for the potential future treatable population for 4D-710, and the potential for redosing. Assessment of transgene expression according to baseline antibody status showed that preexisting antibodies did not affect CFTR transgene RNA or CFTR protein expression. No significant differences in transgene or CFTR protein expression were observed in participants with preexisting anti-A101 capsid antibodies or A101 specific T-cells, compared to those who were negative prior to dosing. Collectively, these data give us continued confidence in our ability to potentially treat all people with CF and also to redose 4D-710 as needed. On this slide, we show that ppFEV1 data from baseline to month 12 for each participant in the two highest dose cohorts.
These participants were enrolled under the original protocol, which mandated standardized follow-up visits for the first 12 months and phone calls between months 12 to 24, so we were not able to get reliable measurements beyond 12 months. The protocol has since been amended to include standard follow-up visits between months 12 and 24, so future participants enrolled will have reliable durability data beyond 12 months. As a reminder, based on data from the pre-modulator era, the average annual rate of percent predicted FEV1 decline for people with CF was approximately 1%-2%, and declines may be greater in people with CF who are ineligible for modulators. In addition, inherent intra-subject variability of FEV1 is approximately 4.5 percentage points.
Finally, small improvements of 2%-4% in percent predicted FEV1 in modulator trials were associated with clinical benefits, including a reduction in pulmonary exacerbations in people treated with Orkambi and Symdeko. Following administration and subsequent transduction of 4D-710, we expect gene expression to be optimized by 3 months post-treatment based on our internal preclinical modeling. Respiratory-related adverse events deemed by investigators as non 4D-710-related, such as pulmonary exacerbations, viral respiratory infections, and hemoptysis occurring within 21 days of a pulmonary function test, are indicated by the circles. In general, you can see lung function remains stable or improved in many cases despite these events. Here, we highlight percent predicted FEV1 data from the 3 participants who had less than or equal to 80% percent predicted FEV1 at baseline and follow-up beyond 6 months.
These participants should be eligible for our potential future phase III trial, for which we have previously reported that we would expect to enroll participants with a baseline percent predicted FEV1 between approximately 40% and 80%. Of note, two of the participants, one from the 1 × 10^15 vector genome cohort, and one from the 2 × 10^15 vector genome cohort, showed improvement in percent predicted FEV1 at 12 months of 5 and 6 percentage points, respectively. We believe these results, if replicated in phase II and phase III, would be compelling support for the use of 4D-710 in the treatment of CF lung disease.
As previously reported, we observed durable improvements in the CF quality of life revised respiratory domain during evaluable time points in the 1 × 10^15 vector genome cohort, showing an average 8-11-point improvement over 12 months, which is consistently above the minimal clinically important difference of 4 points. This finding means that participants treated with 4D-710 in this cohort are consistently feeling better from a respiratory standpoint than they were prior to therapy. The frequency of respiratory adverse events in the 2 × 10^15 vector genome cohort confounded interpretation of the quality of life results. We plan to share additional quality of life data for future enrolled cohorts as it becomes available in our next update. I'll now hand the presentation back to Dr. Cohen to discuss the summary of the data and next steps. Dr. Cohen?
Thanks, Dr. Taylor-Cousar. Now, we would like to summarize our results to date across the 4 dose cohorts and framework for selecting our phase II dose. On the left in blue, we show our dose selection criteria, which focuses on measures of expression, safety and tolerability, and clinical activity. The target profile is shown in blue text, which is based on medical and scientific literature review and consultation with our principal investigators and membership from the CF Foundation. As seen in the 1E15 dose cohort, we achieved all intended dose selection criteria. In the 5E14 dose cohort, we also have results for 1 of 2 currently enrolled participants. Full results following enrollment of participant 3 will be forthcoming and will inform future decisions. Now, we show the results of the highest and lowest dose cohorts, as previously discussed.
Based on the totality of the safety, biomarker, and clinical activity data to date, we have selected 1E15 as our first intended dose for phase II dose expansion. In parallel, we continue to evaluate 5E14 in phase I as a potential second phase II dose. In summary, we're pleased with the emerging profile for 4D-710, based on the phase I results to date, and are nearly complete with dose exploration. We have achieved our objective of generating enough data to make an informed decision to move 4D-710 as quickly as possible into phase II development, in order to reach people with CF who currently have no other disease-modifying therapeutic options.
To summarize our next steps, we plan to begin enrollment with the Phase II dose expansion stage in the second half of 2024, starting with the 1E15 dose and up to a total of 9 evaluable participants. In parallel, the third participant in the 5E14 cohort in Phase I will be enrolled shortly. In addition, as part of this next phase of development, we have recently submitted a protocol amendment for the AEROW Phase I/II clinical trial to our colleagues at the CF Therapeutic Development Network to do the following: Number one, enroll people with CF with lower baseline percent predicted FEV1, down in a range of 50%-90%, and no longer exclude those with frequent historical pulmonary exacerbations, with the objective of more optimally targeting representative participants with mild to moderate pulmonary disease.
Second, introducing a second lung biopsy at 12 months to better characterize transduction, expression, and durability at these lower doses, and the potential timing of redosing if needed. Third, to open a cohort for administration of 4D-710 in participants on CFTR modulators with persistent moderate to severe lung disease, to better understand the safety and potential additional clinical benefit of 4D-710. We expect to begin enrollment in this cohort in the second half of 2024. We anticipate providing an interim data update in mid-2025, after we have completed enrollment and have sufficient follow-up data in all of these important study cohorts. Lastly, the initial GMP-ready suspension manufacturing process was completed in-house at 500-liter scale, and technology transfer initiation to a commercial CDMO is anticipated in the first half of 2025.
We continue to have regular discussions with the FDA, EMA, and CF Foundation regarding the registration path for 4D-710 for treatment of CF lung disease. Based on conversations to date, looking forward to options for potential approval, we anticipate a phase III randomized controlled study with opportunity for crossover, enrolling approximately 60-80 people with CF with lower baseline percent predicted FEV1 lung function, anticipated in the range of 40%-80%. Clinical endpoints would include changes in percent predicted FEV1, quality of life as measured by CFQ-R, and frequency, number, and severity of pulmonary exacerbations. Given the high unmet medical need in the CF population, an accelerated approval path may be feasible to reach these individuals even sooner. We continue to collect additional data from the AEROW study to characterize the correlation between CFTR expression biomarkers and percent predicted FEV1 changes.
Based on the data, we will continue discussions with the FDA and CF Foundation as we expand participant enrollment and generate more relevant participant-level data to better inform us on the potential to pursue an accelerated approval pathway. Depending upon emerging data, we currently expect initiation of a phase III clinical trial in the second half of 2025. We are excited to continue advancing our pulmonology pipeline built around our A101 capsid. This slide highlights the expected data updates over the next 18 months. In addition to the data updates expected mid-2025 for 4D-710, after we complete enrollment and follow-up in phase II, we are looking forward to providing a program update later this year on the 4D-725 program for alpha-1 antitrypsin deficiency lung disease, which is currently in IND-enabling studies.
Now, we'd like to acknowledge and thank our trial participants, their families, our principal investigators and study staff, and the Cystic Fibrosis Foundation Therapeutic Development Network. We'd like to further thank the CF Foundation for their continued guidance, unwavering commitment to clinically relevant research on behalf of people with CF, and their financial support for our lung delivery programs and for 4DMT as a company. I'd now like to hand it back over to David for closing remarks.
Thanks, Alan. I'd now like to conclude by highlighting our key upcoming corporate milestones. We have a strong cash balance sheet with $589 million in cash and equivalents as of the end of Q1 2024, that we expect to give us runway into the first half of 2027. This will allow us to execute on these key near-term milestones. Before I open up the call to questions, I want to take a moment to thank the entire 4DMT team for their remarkable innovation, execution, and dedication. I'd also like to deeply thank our investigators, our clinical trial staff, the CF Foundation, patients, and their families. Thank you, and I'm now happy to open up the call to questions. Operator?
We will now begin our Q&A session. If you have a question, please use the Raise Hand function at the bottom of your screen, or if you have dialed in, please press star nine to enter the queue. Once your name has been announced, you may unmute and ask your question. We will now pause for a moment to assemble the queue. Our first question comes from Salveen Richter with Goldman Sachs. Your line is now open.
Hi, this is Lydia on Salveen. Thank you so much for taking the question, and congrats on the data. Can you just discuss the rationale behind the decision to move forward with the lower dose, understanding that you're continuing to value the higher dose as well, given the positive safety signal that we saw at the higher dose? Thanks so much.
This is David Kirn, CEO and co-founder. Thanks for the question. The rationale is that, yeah, we clearly have strong support for the 1E15 dose to take into phase II, and we just felt we didn't have enough data yet to see whether the 5E14 could be equivalent or superior. So I think we're leaving our options open there, but we're very excited about the 1E15 dose and its potential in phase II and phase III. Thanks for the question.
Thanks, guys.
Our next question comes from the line of Gena Wang with Barclays. Please unmute your line and ask your question.
Thank you for taking my questions. I have a few. First, you know, for the two patients that show improvement, I'm wondering, is there any correlation with the protein expression? And also, regarding the protein expression, I know you showed immunohistochemistry measurement. Have you done the Western blot or mass spec? I think those will be much better quantification in terms of percentage of protein expression improvement. And then lastly, quickly, regarding the potential accelerated approval path versus, you know, the phase III, maybe give a little bit more thoughts, you know, regarding the baseline cutoff there, I think, or 50-90 versus 40-80 FEV1 baseline.
Thanks, Gena, for the question. I'll answer the mass spec question and then turn over the clinical questions to Jennifer and Alan. In terms of the biomarkers, we're thrilled with the robustness of the immunohistochemical staining tests. We've done every imaginable control to show how specific and quantitative that measure is. That having been said, we are looking to add mass spec on in addition later in development, if at all possible, so we're working through the technical approaches to that test as well. But we think this is by far and away the most robust histologic assessment that's ever been done in CF patients in terms of our ISH and IHC. So, Dr. Taylor-Cousar, would you like to speak to the FEV1 responses and cutoffs?
Yeah, great question. So in terms of the protein expression, if you think back to that slide, the protein expression was quite robust at all dose levels, so there wouldn't really be a correlation, a differentiation in the correlation between different dose levels in terms of SB1, especially given our very small sample size in this phase I part of the study. And in terms of the cutoff-
... It's always critical to show that a new drug is safe in people with higher lung function before moving down into lower lung function, if that's really the consideration about how we'll think about the phase III data is continuing to show safety in this group of people with a little bit higher lung function before we move into lower lung function groups.
Thank you. Alan, anything to add to that?
No, I think Dr. Taylor-Cousar covered it very well. Thank you.
Thank you.
Thank you.
Our next question comes from the line of Tazeen Ahmad with Bank of America. Please unmute your line and ask your question.
Hi, can you hear me?
Yes. Yes.
Hello? Okay, great. A few for me as well. So the 10^15 that you've decided to move forward with, is it that all 9 patients are gonna be dosed to that level, or do you have any opportunity to dose higher with some of those patients? And then by the time you reach the middle of next year, when we expect to have this interim read, will any of the patients have reached 12 months and have had biopsy? And then lastly, just based on the data you've collected so far, just wanted some more clarification about the path to accelerated approval, especially for the patients who don't have any other options currently. What has FDA indicated, if anything, on the minimal threshold of patient number or level of efficacy that would be needed to support such a path? Thanks.
Thank you, Tazeen, and I'll give some intro remarks and then hand it over to Dr. Cohen and Dr. Taylor-Cousar. In terms of the development path here, yeah, we're very pleased with the 1E15 dose level. We think the 2E15, we're really, at that point, just saturated the airways and starting to get transduction of the interstitium, which is not what was in our target profile. So, we'll be at the 1E15 for the additional nine patients. And then again, if the 5E14 continues to evolve and emerge as a very promising dose, then we can consider an amendment to add additional patients at that dose level. So we'll keep an eye on that as we add the third and final patient and get more follow-up.
Alan, do you want to speak to the accelerated approval question and also just the timing of how much data we'll have middle of next year?
Sure. Not a problem, David. So, we're currently amending the protocol after conversations with the CF Foundation and the Therapeutic Development Network to add the second biopsy, and we're excited about that for the obvious reasons. We want to be able to better assess the durability and continued transfection out at least a year. We will have that protocol amendment in shortly, and it's quite possible that we may have at least one subject with paired biopsies by that time. However, it would be speculative to say for sure that that would be the case. In terms of accelerated approval, I think the approach here would be to base it really on emerging data in these lower dosing cohorts. I think part of your comment asked about going up from 1 × 10^15.
Right now, we currently believe that among the four patients in that cohort, given that we observed the safety effects and had enough of a safety signal to suggest that that dose is no longer worth pursuing, our intention here would be to go down, if at all, and to look at the lowest effective dose as per FDA guidances. So right now, we feel very comfortable with the 1E15 dose.
We're going to complete filling the 5E14 cohort with a third subject in the coming weeks, and then essentially follow up with a repeat biopsy, a primary biopsy at 12 months, and then continue to monitor that patient and additional patients we'll be adding to these two lower cohorts over the next 6-12 months and share that data as we have it. The accelerated approval would really be based on the totality of the emerging data, and speculation as to what would constitute a sufficient package for that right now, I think, would be too premature.
Dr. Taylor-Cousar, would you like to add anything?
Nope. I think Dr. Cohen covered it well. Thank you.
Our next question comes from the line of Ryan McElroy with Leerink. You may unmute and ask your question.
Thanks for taking our question. I'm on for Mani. We just had two quick questions. One for baseline FEV. Can you provide any detail? Is this determined by a serial measurement or more of just a single measurement at a, you know, on a single day? And then second question, just wondering, why do you guys believe kind of the robust transgene expression is, is not translating necessarily to a clinical benefit as, as you'd probably expect? Thank you.
Well, I'll start out and then Alan can weigh in. We do think it's clearly leading to clinical benefit. If you look at the 1E15 dose level, and the quality of life benefit at all time points, far, far in excess of the minimal clinically important difference, we have a very robust quality of life data set at that dose level, despite all the intercurrent illnesses that these people were getting as they came out of COVID. I think we're also thrilled to see two out of the three patients who are in that range of mild to moderate lung disease showing FEV1 responses that are, again, very clinically meaningful. So we apologize if we didn't make it clear that there's clear clinical benefit that was emerging from this phase I study.
Jen, do you wanna weigh in on that?
... Yeah, I, I agree with you, David. I think we have seen some clinical response. The other thing that I would say is that we're, again, talking about very small numbers. If you look at the data from the triple combination modulator therapy, there's a wide range of response. You wouldn't expect every single person to have the same response. The fact that we have response into the small number of people, I think is very promising.
Additionally, just to answer your other question about the baseline measures. You know, obviously, these CF patients are meticulously managed and cared for, and they have long historical records of what their pulmonary functions are on any given day, at every given month, at every given visit. At the study point where they're screened, we look at the screening results as well as the enrollment day results, and are looking to see fairly consistent reproducibility from those two points. But we do have to draw a line in the sand for collecting data. So the baseline data is not an accumulation of multiple days of testing, but rather using standard ATS/ERS guidelines, where you need to see three reproducible performances of spirometric measures within the acceptable range, and then the values are ascertained using those guidelines.
We're using standard ATS/ERS practices for our baseline measures and then all subsequent measures. We're also using consistent equipment provided to each of the sites with centralized reading. We're trying to make those measures and that data as tight as possible.
That's helpful.
The only thing I would add-
And one more, if you don't mind. Oh, go ahead. Sorry.
Sorry, I was just gonna add that on the day of dosing, we do do serial measurements, so maybe that's what you're thinking of, just to make sure that there's no evidence of acute reactivity. And we have not seen that with the serial measurements, but that's on the day of dosing.
Very helpful. And one last thing, as we can kind of consider further data updates into the future, is it reasonable to expect that we will not receive any FEV1 data for these patients into the future? Or is, as you mentioned, the trial amendments or at-home visit measurements going to be an option in the future? Thank you, guys.
I'll just start, and then Alan can weigh in. Again, we're thrilled that we're seeing these FEV1 results, and in particular, at 12 months. I mean, this is not at, you know, a week or 2. This is very clinically meaningful out to 12 months and would be an approvable endpoint with, you know, based on our interactions with FDA. Alan, do you want to speak to further follow-up beyond that?
Yeah, sure. Thank you, David. So the original protocol, it's worth noting that the CF Foundation Therapeutic Development Network is very protective, appropriately so, of the CF patient community, and do not want to burden them with too much excessive testing or elongated studies, which may preclude their inability to participate in additional research or therapies as they emerge. That said, the original protocol actually limited our ability to collect data on those subjects through a year. We've since extended that timeframe for two years. So to answer your question, we will be continuing to collect data in subsequent cohorts through two years, not using home equipment, but actually referencing back to the primary investigator site, using the same equipment, same techniques, with same central reading, moving forward.
Many of the patients in the last cohorts that we've enrolled are enrolled in the two-year elongated study. Additionally, given that the FDA, with gene therapy, is expecting upwards to 5 years of longer-term follow-up, we are further amending the protocol for years 3-5, to continue to collect historical data. It will be done less rigorously if patients are unable to continue to return to their primary investigator site. We're making every effort possible to try to make this manageable for patients, understanding that 3-5 years may be a large expectation for many of them.
Our next question comes from the line of Yun Zhong with Jefferies. Please unmute and ask your question.
Excuse me. Good morning. Thank you very much for taking the question. And this is for Kelly. And so one question is, did you measure the antibody production after treatment? Would that information be required for your discussion with the FDA on redosing, or is the observation that pre-existing neutralizing antibody had no effect on the protein expression be sufficient? And the second question is a follow-up question on dose selection. Did I see that on one slide that you showed intensity of CFTR expression was actually higher at the dose of 4E15... Sorry, 5E14? And I believe that you said the third patient was gonna be the last patient.
How comfortable are you that only three patients will give you sufficient information to convince them that 1E15 will be a superior or better dose than 4E15?
Thank you for the question. I'll kick it off to Alan, and Dr. Taylor can weigh in. You know, as I said, we're very confident in the 1E15 dose. We're seeing FEV1 effects, we're seeing quality of life improvements, and very strong biomarker data. So we're very comfortable with that dose level. I think with these small numbers, we're not gonna be able to say whether 5E14 is better, but we certainly can say so far, it matches our target profile for a phase II or phase III dose, and we'll continue to follow all 3 patients from that dose level over time and see whether we want additional phase II data at that dose.
So I think it's still certainly a promising dose level that we may explore in phase II based on phase I data. Alan, do you want to address some of the other questions about the protein staining?
Yeah, I'm not sure what you understood on the presentation just moments ago, but I think the points of the graphics in the presentation that Dr. Taylor-Cousar gave was essentially highlighting the fact that we're seeing quite generous staining and protein production across all four doses thus far. And that there was starting to be a lower preponderance of protein staining or IHC predominance as we went from the highest to the lowest doses, but it was only a small amount. Most notably, we do believe that we were seeing less prominence of staining in the interstitium, which we believe is a place that not only were we not targeting, but which we don't believe is necessary moving forward.
As such, that's why we're stopping enrollment and consideration of the 2E15 dose and are looking at the lower doses, most notably 1E15 and 5E14. Also, given the preponderance of messenger RNA that we've seen there, and the magnitude of... and the linear progression of the dosing there, we believe that the sweet spot right now for messenger RNA, which we believe may be a more sensitive measure for us right now, falls somewhere between the 1E15 and the 5E14 dose.
As David said just a moment ago, I don't think we have enough patient data yet, now that we're getting ready to enroll subject number 3 in the 5E14 dose, and enough time hasn't passed for us to really make a determination as to whether or not one or the other dose might be optimal for advancing into phase III. I think right now it's a dose de-escalation, which is unusual, but we're happy to do it. But most importantly, we're gonna need to let the data that we collect and the tolerability, safety, and efficacy data that we generate, drive decision-making moving forward. I think we just simply need a few more subjects over a longer period of time to make that determination comfortably.
Now, I didn't have any question on the-
The only thing I would... Oh, sorry. I was just gonna add one more thing. To answer very specifically that question about the slide regarding the % positive for protein expression. So if you look at the CFTR % positive epithelial cells, that's actually quite equivalent between the different dose groups. You might have seen that the intensity looks higher in the 5 × 10^14 vector genome dose, but you have to remember, that's only from 2 samples compared to the other groups. So again, if you think about the range of response, we don't really have enough samples to make a true determination of what that dose level looks like in terms of intensity.
Thank you. And, another question was on the, neutralizing antibody, please.
Yeah, so that's something we're collecting over time, and we'll report that at a later date. But we certainly, based on animal model data and other human data, would expect antibodies to increase after dosing. But again, we believe there's strong animal data, and now human data to suggest we can redose despite those antibodies. We think that the reason is that the concentration of the vector is so high in those droplets that are distributed throughout the airway by the AeroEclipse II device, which is a really beautiful device for essentially blanketing the airways.
That those droplets come in direct contact with the cells that need to be transduced, and with this vector evolved for resistance to preexisting antibodies and then high degree of transduction efficiency, we get very we believe we get very rapid uptake into the cells, and, and therefore, antibodies would not be an issue. Thanks for the questions.
Thank you.
Our next question comes from the line of Jon Miller with Evercore. Please unmute and ask your question.
Hey, guys, thanks for taking my question, and congrats on all the data. I would also like to ask a question on the go-forward dose that you've picked, and maybe coming from the other direction. Given that it seems like even the very lowest dose that you've looked at delivers supraphysiological CFTR protein levels, I guess, are you going with 1 × 10^15 just because you have the safety coverage there already? Or is there a clear expectation that that dose is going to perform better than these lower doses that are still at supraphysiological CFTR?
Thanks, Jon, for the question. Jen, do you wanna answer the question?
Yeah. So I think that the first answer is that we do already have safety data in that first cohort. People tolerate it quite well. And we also have efficacy data, both in terms of FEV1, with a participant having an improvement that was clinically significant in their FEV1, as well as sustained improvement in their quality of life scores that are, again, well above the minimally clinically important difference. So I think that's the main reason we're looking at this dose. Now, if we see clinical outcomes that are equivalent with these lower doses, of course, again, as Alan said earlier, you want to use the lowest dose that you possibly can to achieve the same efficacy. But right now, we are seeing efficacy at the 1 × 10^15 vector genome dose, and that's the major decision.
I don't know if you want to add anything, Alan?
Yeah, I think just one more thing. You know, this is a balancing act of trying to titrate a tolerability, efficacy signals, and of course, the one thing we haven't spoken about much is durability. And undoubtedly, the higher doses will lead to longer durable signal and treatment, perhaps elongating the dosing intervals between subsequent dosings much longer. So I think we have to be sensitive to that as well. Right now, I think we're thinking about a dosing interval of something like 1.5-3 years, possibly. And I think keeping that in mind when we're looking at, in particular, the second biopsy samples that we're going to start collecting shortly, will be enormously informative in terms of helping to determine the doses moving forward.
Great. Thank you. And then maybe one on the modulator combo population that you just started talking about. Could you maybe remind us how you're defining moderate to severe residual lung disease on modulators? And maybe speak a little bit to the decision to go to the functionally defined population rather than a genetically defined population, or could you relate those two things together? Are there specific genotypes that you expect are gonna be falling into this residual lung disease population, or is it purely functional?
Thanks, Jon. Jennifer?
Yeah. So what we're trying to find are those people who still have an FEV1 that's lower than 70%, so they still have room for improvement in their lung function, and those who are also continuing to frequently have pulmonary exacerbation. So those are the group of people who still need antibiotics, are still taking a lot of other CF therapies. So there are simplified trials that suggested that for people who have very good lung function and who are on modulators, you could stop some of the other symptomatic treatments. But for that group who's under 70, they're still having that huge treatment burden and sometimes having to be admitted, oftentimes having to get oral antibiotics. So that's the group that we're really targeting and saying that they have an unmet need.
Thank you.
Our next question comes from the line of Kostas Biliouris with BMO. Please unmute and ask your question.
Hello, everyone. Thanks for taking our question, and congrats on the progress. Maybe one question for Dr. Taylor on the impact of baseline on the ability to show FEV1 improvement. A couple of data points. There is a study in pediatrics, which shows that in some children, even with a 90% FEV1 baseline, you still see a 10% improvement on the first 15 days, which kind of suggests that even with a very high baseline, you can still see some improvement. And the second data point is that I believe FEV1 can go even beyond 100%, because it's just a relevant endpoint there to some particular population.
Can you talk about your thoughts around the patients who have high baseline, and what would prevent these patients from improving even further, beyond 90% or even beyond 100%? And then I have one more follow-up. Thank you.
Sure. No, it's a great question, and I think you do have to consider the degree of structural lung disease that's present in many adults versus those in children. So whether or not we've met a ceiling with FEV1 improvement in people with higher lung function, that's yet still to be determined. We don't have any CFTR therapy at this point that corrects to wild type. And until we do, I don't think we know what the ceiling is on improvement in FEV1, but I do think that it is more difficult to improve percent predicted FEV1 in people with more severe structural diseases, not always really well reflected in FEV1.
Okay, thank you. And maybe one follow-up. I think you touched on already, but I think you saw a dose dependence, increase in the mRNA of CFTR, but you saw no dose dependence on the CFTR protein between 1 × 10^15 and 2 × 10^15. So can you explain the difference between the mRNA dose dependence and protein, dose independence there? Thank you.
Sure. I mean, I can start, and Alan may want to weigh in as well, but I think that part of it is that we're reaching saturation with protein expression. So really, we're above 99%, essentially, with all of it. And so it's hard to show a difference between doses when you're reaching saturation. But Alan, I don't know if you want to weigh in as well.
Yeah. I mean, I think right now, at least in our hands, what we're seeing is that the linear differences that we're seeing when we're measuring messenger RNA seem to be much more sensitive from a dose de-escalation standpoint than the oversaturated situation we're finding ourselves in with the staining for CFTR protein. So, right now, also, let's not forget that the samples that we're looking at, as Jennifer highlighted a moment ago, some of those lower doses, we really only have two or three biopsies from a single patient.
So once again, I think this becomes a question of needing to enroll a few more patients, get more informative biopsies and cell brushing samples, and increase the pool of material that we have to draw from, and then make determinations based on more data over longer periods of time. But right now, I think messenger RNA is proving to be a much more sensitive measure for us, and I think it will help guide us as we move forward.
Thank you. Very helpful.
At this time, I'd now like to turn over the call to Dr. David Kirn for closing remarks.
Well, thank you, everyone. We're really excited with this data update and the efficacy that we're seeing and the tolerability of the 1E15 dose level. So we're excited about phase II, and I'd just like to thank the 4D team, including Dr. Alan Cohen, our SVP of Pulmonary and head of this program, and in particular, Dr. Taylor-Cousar, for all of your tireless work and dedication to patients and to this trial, and for calling in from all the way from Glasgow today. Thank you all of you for your time and interest today.