1. What do the new results on NFL-101 show?

NFL Biosciences presented new efficacy analyses from the Phase 2 CESTO II study. These analyses identify a target population of patients carrying a biomarker defined by baseline NFL-101-specific IgG1 levels below 200 ng/mL before treatment. This population represents 57.2% of the patients analyzed.

In this target population, NFL-101 showed enhanced efficacy versus placebo. In other words, compared with the general population, efficacy in the target population is higher, the effect size versus placebo is greater, and statistical significance versus placebo is reached over the 4-week period corresponding to the primary endpoint, using both confirmation methods: urinary cotinine and exhaled CO. These results support the continued development of NFL-101 in a personalized medicine approach, which will need to be prospectively confirmed in the next clinical study.

2. What is the biomarker-positive target population?

The target population corresponds to patients whose baseline NFL-101-specific IgG1 level is below 200 ng/mL before treatment. In CESTO II, 306 of the 318 patient samples were analyzed, and 57.2% of them presented this profile.

These patients have an immunological profile that is more receptive to the action of NFL-101. Conversely, biomarker-negative patients have a less receptive immunological profile. Efficacy in these patients was found to be comparable to that of placebo.

3. What are IgG1 and specific IgG1?

IgG1 are a subclass of antibodies associated with acquired immune responses and immunological memory. NFL-101-specific IgG1 reflect the immune memory developed over time through chronic exposure to tobacco.

As NFL-101 is composed of tobacco leaf protein extracts, these antibodies indicate prior recognition of these antigens by the immune system. Their baseline level therefore provides information on the patient’s pre-existing immune status with regard to tobacco antigens.

4. What does the IgG1 < 200 ng/mL threshold mean?

The 200 ng/mL threshold corresponds to the concentration used to distinguish biomarker-positive patients from biomarker-negative patients. “ng/mL” means nanograms per milliliter, a concentration unit used to measure antibody quantities in a biological sample.

Patients with low NFL-101-specific IgG1 levels would retain immune receptivity allowing NFL-101 to act fully. Conversely, patients with higher levels would already have an established or more tolerogenic immune response, associated with lower receptivity to treatment.

5. How should the 4-week and 12-month results be interpreted?

When the protocol was designed, the number of patients to be included was calculated based on efficacy assumptions for 4-week continuous abstinence. This 4-week abstinence period was defined as the study’s primary endpoint, meaning the endpoint on which statistical significance had to be demonstrated for the study to be considered successful. The other continuous abstinence measures, up to 12 months, are secondary endpoints designed to document the persistence of the treatment effect. As the number of patients to be included was not defined based on these endpoints, statistical significance was not expected for them.

For both the 4-week and 12-month endpoints, compared with the general population, efficacy in the target population is higher and the difference versus placebo is greater. In addition, for the 4-week endpoint, statistical significance versus placebo is reached using both validation criteria: urinary cotinine and exhaled CO.

6. Does the absence of statistical significance at 12 months call the results into question?

No.

CESTO II was designed to demonstrate efficacy on 4-week continuous abstinence, which was the protocol’s primary endpoint. Continuous abstinence at 12 months was a secondary endpoint. The number of patients, calculated for the primary endpoint, was not sufficient to demonstrate a statistically significant difference at 12 months, as continuous abstinence naturally decreases over time.

For the 12-month endpoint, compared with the general population, efficacy in the target population is higher and the difference versus placebo is greater. Ultimately, an efficacy rate of 21.3% at 12 months and a 2.4-fold efficacy ratio versus placebo are excellent results, demonstrating the value of selecting this target population.

7. Why is this referred to as a post-hoc analysis?

It is referred to as a post-hoc analysis because the biomarker was not predefined in the initial CESTO II protocol. NFL-101-specific IgG1 were analyzed as part of the work to better understand NFL-101’s mechanism of action, and the 200 ng/mL threshold was then identified based on the study data.

This does not make the analysis irrelevant. It simply means that the biomarker was identified during an exploratory and biological understanding phase. To become a fully robust tool in clinical development, it will need to be prospectively confirmed in a future study, with a threshold and use defined in advance.

8. What does prospective confirmation of the biomarker mean?

Prospective confirmation means testing the biomarker in a new study designed from the outset to use it. In practical terms, the specific IgG1 threshold and biomarker-positive patient status would be defined before patient inclusion and analysis.

NFL Biosciences plans to integrate biomarker-positive patient status as an inclusion criterion in the protocol of the next NFL-101 clinical study. This step is important because it must confirm that the relationship observed in CESTO II can be reproduced in a pre-specified study.

9. Has the biomarker already been validated?

The four evaluation criteria are met in the available analyses: enhanced efficacy in biomarker-positive patients, comparable placebo response between subpopulations, limited effect in biomarker-negative patients, and biological consistency with the mechanism of action.

However, regulatory validation of a biomarker requires prospective confirmation. The most precise wording is therefore that the biomarker is supported by coherent and promising data from CESTO II, and that its prospective confirmation is planned in the next clinical study.

10. Why compare NFL-101 with Champix if the comparison is indirect?

Because, to date, an indirect comparison is the only way to assess the potential of NFL-101 versus existing treatments. NFL Biosciences presents the comparison with the EAGLES study as an indirect comparison, intended to provide positioning evidence. It does not constitute a direct comparative study between NFL-101 and Champix.

This comparison nevertheless makes it possible to position the results observed with NFL-101 against published reference treatments. NFL Biosciences notably emphasizes the comparison at 3 months after the end of treatment, a period considered more relevant in the context of very different administration regimens. This interpretation should remain cautious and does not replace a direct comparative study.

11. What is the difference between the general population and the target population?

The general population refers to all patients without prior biomarker-based selection. The target population refers to biomarker-positive patients, i.e. those whose baseline NFL-101-specific IgG1 level is below 200 ng/mL before treatment.

In CESTO II, NFL-101’s efficacy is higher and the difference versus placebo is greater in this target population than in the general population. This observation has led NFL Biosciences to consider developing NFL-101 through a personalized medicine approach.

12. Is NFL-101 being replaced by NFL-102?

NFL-102 does not replace NFL-101. The two drug candidates are being developed in parallel with different positioning: NFL-101 in a personalized medicine approach, focused on a biomarker-positive target population; and NFL-102 in the general population of smokers, with an expanded mechanism of action.

The two products have complementary positioning and different mechanisms of action.

The development priority for advancement into Phase 3 will depend on efficacy results, regulatory discussions, discussions with potential partners and, where applicable, resource allocation.

13. What is the difference between NFL-101 and NFL-102?

NFL-101 is a tobacco leaf protein extract designed to act primarily on immunological and neuroinflammatory mechanisms. Its development is now being considered in a target population carrying a predictive efficacy biomarker.

NFL-102 contains the components of NFL-101 as well as additional compounds derived from tobacco leaves. It targets an expanded mechanism of action, notably involving signaling pathways implicated in the deeper mechanisms of addiction, and is being developed for the general population of smokers.

14. What is the role of the TONIC study?

TONIC is the planned Phase 2 study designed to evaluate NFL-102 in the general population of smokers. Its objectives are to confirm safety, assess efficacy and select the optimal dose of NFL-102.

The study is expected to include 450 participants randomized in a double-blind, placebo-controlled design, in France, across approximately ten clinical centers. The planned primary endpoint is 4-week continuous abstinence, confirmed by urinary cotinine, with a final visit at Day 43. A product safety assessment will be carried out by an independent committee after the first 40 patients.

15. When could Phase 3 start?

The objective is to advance at least one of the two candidates into Phase 3 by 2027-2028. For NFL-101, the next step is to engage with regulatory agencies on the biomarker and the clinical design of the Phase 3 study. For NFL-102, the priority is the manufacturing of the clinical batch, the submission of the TONIC study application and the availability of the study results.

The effective start of a Phase 3 study will therefore depend on regulatory feedback, the results of TONIC, the prioritization decision between the programs and financing or partnership conditions.

16. How could the next clinical phases be financed?

NFL Biosciences will prioritize financing Phase 3 studies with pharmaceutical partners, through licensing agreements, co-development agreements or other possible formats.

Self-financing would be an alternative scenario, requiring the Company to raise additional resources when financing Phase 3.

17. Where do discussions with potential industrial partners stand?

The Company is in discussions with several potential partners, with possible formats including global agreements or agreements by geographic region. These discussions remain confidential for the time being. The NFL-101 results in the biomarker-defined population have not yet been presented.

There is no firm timetable for concluding a partnership. The timing and terms of any potential agreement will need to be assessed in light of the interests of the Company and its shareholders.

18. What is the role of McLean Hospital?

McLean Hospital has a twofold role: first, McLean is conducting additional work on the mechanism of action; second, Prof. Scott Lukas plays a key scientific opinion leader role, particularly in supporting the value of the results, publications and conference presentations.

19. What does the biomarker patent application change?

The patent application aims to protect the use of the predictive biomarker associated with NFL-101, in particular the use of specific IgG1 to identify a population of patients more likely to respond to treatment. It also covers the potential development of a test to identify biomarker-positive patients.

This patent application strengthens NFL Biosciences’ intellectual property around the tobacco franchise. The Company now holds five patent families in smoking cessation based on tobacco extracts.

20. How could the biomarker test work in practice?

The process would be based on a blood sample taken before treatment. The physician would prescribe a test measuring NFL-101-specific IgG1. If the result is below 200 ng/mL, the patient would be considered biomarker-positive and potentially eligible for NFL-101.

This type of test could be performed in a community or hospital laboratory. Treatment with NFL-101 is based on two subcutaneous injections administered one week apart, on Day 1 and Day 8. This pathway remains linked to clinical development and will need to be confirmed as part of the next regulatory steps.

21. What are the main points to be confirmed?

The results presented strengthen the development strategy for NFL-101 and provide a solid basis for Phase 3 preparation. Discussions with regulatory agencies will focus in particular on the prospective validation of the biomarker, as well as on the inclusion population and the sizing of the study.

Other elements also remain to be clarified: the relative development trajectory between NFL-101 and NFL-102, which will depend on the results of the TONIC study, the financing conditions for Phase 3 studies and the potential conclusion of agreements with industrial partners. These elements are not weaknesses to be concealed; they correspond to the normal next steps in biotech clinical development.