Abstract: Introduction Failure to respond to steroid therapy for intestinal acute graft-versus-host disease (aGvHD) is associated with limited further therapeutic options. Fecal microbiotherapy is defined as the perfusion of treated stool from one or several healthy donors via the upper or lower gastrointestinal (GI) route aiming at improving microbial diversity and functionality. Here we report clinical outcomes from a 76-patient cohort with steroid refractory (SR) GI-aGvHD treated with the pooled allogenic fecal microbiotherapeutic MaaT013. Twenty-four patients were treated in the prospective, single-arm, phase IIa, HERACLES study (NCT03359980) while 52 patients were treated in an expanded access program (EAP). Patients and methods For HERACLES, 24 patients with grade III-IV SR-GI-aGvHD were treated with MaaT013 in 26 European sites, as a 2 nd line therapy after SR diagnosis and evaluable for treatment response. In EAP, 52 patients with steroid-dependent or SR-GI-aGvHD (classical n=41, late onset n=3, overlap syndrome n=8) were treated. These patients had previously received and failed 1 to 6 lines (median 3; 40/52 received ruxolitinib) of GvHD systemic treatments. GI-GvHD response was evaluated weekly and 28 days after day (D) 0 (inclusion for HERACLES or 1st dose for EAP). For all patients, GI-overall response rate (ORR) at D28 was defined as the proportion of patients achieving complete response (CR), very good partial response (VGPR) or partial response (PR), compared to D0, without the use of additional systemic therapy. Other endpoints included the best overall response (BOR) achieved at any time, and overall survival (OS). Prepared under GMP, MaaT013 is characterized by a highly consistent richness of 455 ±3% OTUs and an Inverse Simpson index & gt; 20. Treatment comprised 3 MaaT013 doses, each composed of 30 g of feces in 150 mL volume of inoculum (total 90 g of feces from 4 to 8 healthy donors) administered by enema (except for 2 EAP patients by nasogastric tube). All patients received at least 1 MaaT013 dose, 92% (HERACLES) and 87% (EAP) at least 2 doses, and 50% (HERACLES) and 71% (EAP) the full treatment course. In HERACLES, the reasons for not applying the 3 rd dose were death (n=5), physician decision to introduce salvage therapy (n=5), and ICU hospitalization (n=2)). Results In HERACLES, the GI-ORR was 38% including 5 CR, 2 VGPR and 2 PR. In EAP, positive GI-response was achieved in 31/52 patients (60% with 16 CR, 11 VGPR and 4 PR). Considering the GI-BOR, 13/24 (54%) and 35/52 (67%) achieved at least a PR in HERACLES and EAP respectively. In HERACLES, OS was 29% at month (M) 6 and 25% at M12. OS was significantly higher in responding (R) patients (achieving at least PR at D28) compared to non-responding (NR) (44% vs 20% at M6 and 44% vs 13% at M12, logrank p=0.047). In EAP, OS was 48% at M6 and 37% at M12, and significantly higher in R patients compared to NR (71% vs 17% at M6 and 62% vs 6% at M12, logrank p & lt;0.0001). In HERACLES, treatment with MaaT013 was characterized by excellent tolerance: 252 Treatment-Emergent Adverse Events (TEAE) were reported for the 24 patients, the majority being infections (79%) and GI disorders (62%), as expected in GvHD patients. Of these 252 TEAE, only 2% (5 serious events in 2 patients) could not reasonably be excluded from being related to MaaT013 by the investigators. Shotgun sequencing in these 5 TEAE revealed that the causative infectious agents could not be detected in the administered MaaT013. In EAP, the safety profile of MaaT013 was considered satisfactory for all patients. 16S microbiome analyses were performed in the HERACLES population and showed that MaaT013 produced an early increase in α-diversity at genus level with a significant increase in Richness index at all evaluated timepoints (p & lt;0.003). At D28, R patients had higher values of α-diversity indices (Shannon p=0.005 and Richness p=0.038) compared to NR patients, and higher proportions of MaaT013-derived species in the total composition of R microbiota (p=0.043). Conclusion We herein report the treatment of 76 SR-GI-aGvHD patients using a full ecosystem, pooled-donor, high-richness biotherapeutic. The D28 GI-ORR was 38% and 60% in HERACLES and EAP respectively and this clinical benefit positively and significantly impacted OS (44% and 62% M12 in HERACLES and EAP R patients respectively). MaaT013 was shown to be safe and effective in these heavily immunocompromised patients, warranting further exploration of this approach. Figure 1 Figure 1. Disclosures Malard: JAZZ pharmaceuticals: Honoraria; Sanofi: Honoraria; Astellas: Honoraria; Biocodex: Honoraria; Therakos/Mallinckrodt: Honoraria; Janssen: Honoraria. Loschi: CELGENE/BMS: Honoraria; AbbVie: Ended employment in the past 24 months, Honoraria; Gilead: Ended employment in the past 24 months, Honoraria; Novartis: Ended employment in the past 24 months, Honoraria; Servier: Ended employment in the past 24 months, Honoraria; MSD: Honoraria. Cluzeau: Agios: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel, accommodations, expenses, Speakers Bureau; Roche: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria, Speakers Bureau; Jazz Pharma: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria, Speakers Bureau; Amgen: Speakers Bureau; Pfizer: Other: travel, accommodations, expenses; Astellas: Speakers Bureau; Takeda: Other: travel, accommodations, expenses. Huynh: Jazz Pharmaceuticals: Honoraria. Holler: MaaT Pharma: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Vehreschild: SocraTec R & D GmbH: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Ferring: Consultancy, Speakers Bureau; Farmak International Holding GmbH: Consultancy, Honoraria, Speakers Bureau; Bio-Mérieux: Consultancy, Speakers Bureau; Basilea: Consultancy, Speakers Bureau; Arderypharm: Consultancy, Speakers Bureau; Alb Fils Kliniken GmbH: Consultancy, Speakers Bureau; Takeda Pharmaceutical: Research Funding; Seres Therapeutics: Research Funding; Roche: Consultancy, Research Funding, Speakers Bureau; Organobalance: Consultancy, Research Funding, Speakers Bureau; Merck/MSD: Consultancy, Research Funding, Speakers Bureau; MaaT Pharma: Consultancy, Research Funding; Immunic AG: Consultancy, Research Funding, Speakers Bureau; Glycom: Research Funding; Gilead Sciences: Consultancy, Research Funding, Speakers Bureau; Evonik: Research Funding; Da Volterra: Consultancy, Research Funding, Speakers Bureau; Biontech: Research Funding; Astellas Pharma: Consultancy, Research Funding, Speakers Bureau; 3M: Research Funding. Gasc: MaaT Pharma: Current Employment. Plantamura: MaaT Pharma: Current Employment. Mohty: Sanofi: Honoraria, Research Funding; Pfizer: Honoraria; Novartis: Honoraria; Takeda: Honoraria; Jazz: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria; Celgene: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria; Astellas: Honoraria; Amgen: Honoraria; Adaptive Biotechnologies: Honoraria.

Abstract: Acute myeloid leukemia (AML) intensive chemotherapy combined with broad-spectrum antibiotics, leads to gut microbiota dysbiosis promoting pathological conditions and an increased incidence of complications. Here we report findings from a phase II single-arm, multicenter study evaluating autologous fecal microbiota transfer (AFMT) in 25 AML patients treated with intensive chemotherapy and antibiotics (ClinicalTrials.gov number: NCT02928523). The co-primary outcomes of the study are to evaluate the efficacy of AFMT in dysbiosis correction and multidrug-resistant bacteria eradication. The main secondary outcomes are to define a dysbiosis biosignature, to evaluate the effect of dysbiosis correction on patient clinical status, to assess the short and mid-term safety of AFMT in this immunocompromised population, and to evaluate the feasibility of the AFMT procedure and acceptability by the patient. Intensive induction chemotherapy induces a dramatic decrease of α-diversity indices, and a microbial dysbiosis with a significant shift of the microbial communities and domination of pro-inflammatory families. After AFMT treatment, α-diversity indices return to their initial mean levels and the similarity index shows the restoration of microbial communities. The trial meets pre-specified endpoints. AFMT appears to be safe and may be effective for gut microbiota restoration in AML patients receiving intensive chemotherapy and antibiotics, with an excellent gut microbiota reconstruction based on both richness and diversity indices at the species level.

Abstract: Introduction Intestinal graft-versus-host disease (GvHD), following allogeneic hematopoietic stem cell transplantation (allo-HSCT), has a high mortality rate and a reduced life-expectancy. In this context, failure to respond to steroid therapy is associated with limited further therapeutic options, thereby representing an unmet medical need. Aiming at restoring microbiome functions, fecal microbiota transfer (FMT) has proved to be a promising treatment modality in this challenging clinical setting, with recent studies reporting favorable results in steroid refractory-acute GVHD (SR-aGvHD) patients. Here we report on the use of a next-gen FMT product "MaaT013", a standardized, pooled-donor, high-richness microbiota biotherapeutic, used to treat 29 patients with intestinal aGvHD as part of a compassionate use/expanded access treatment program. Patients and methods Twenty-nine allo-HSCT recipients with steroid-dependent or SR intestinal GvHD (classical aGVHD n=22, late-onset aGVHD n=2; aGvHD with overlap syndrome n=5) were treated with MaaT013 biotherapeutic as part of a compassionate use/expanded access treatment program of the developer MaaT Pharma. These patients had previously received and failed 1 to 5 lines (median 3; 22/29 received ruxolitinib) of GvHD systemic treatments. Each patient received 1 to 3 doses (median: 3; total doses administered: 71) of MaaT013, a 150 mL bag, by enema (n=28) or nasogastric tube (n=1). GI-GvHD response was evaluated 7 days after each administration and 28 days after the first dose. Prepared under Good Manufacturing Practices, MaaT013 biotherapeutics are characterized by a highly consistent richness of 455 ±3% Operational Taxonomic Units and an Inverse Simpson index greater than 20. Batch release specifications are based on potency (viability), identity (diversity), and purity (microbiological safety testing and proportion of proinflammatory species), ensuring the desired consistency across batches. Results We observed a GI overall response rate of 59% (17/29) at day 28 after first dosing, including 9 complete response (CR), 6 very good partial response (VGPR), and 2 partial response (PR). Considering the best GI response achieved, 20/29 patients (69%) achieved at least a PR, with 9 CR, 8 VGPR and 3 PR. Among the 29 treated patients, 16 were still alive at last follow-up (median follow-up (FU): 131.5 days; range [28; 413]) and 6 months overall survival was 54%. Among the 9 patients achieving CR, all were still alive at last follow-up (median FU: 197 days; [49; 301] ) and were able to taper or stop steroids and immunosuppressants. Among these 9 patients, three patients presented GI symptom recurrence between 2 and 3 months after dosing. In 2 patients, this recurrence occurred after heavy antibiotic treatment, one of them received an additional MaaT013 dose and achieved a second CR. Of note, among these 9 patients, mild chronic mucosal GvHD symptoms persisted in one patient, and molecular relapse of hematologic malignancy was observed in another. The safety of the MaaT013 microbiota biotherapeutic was satisfactory for all patients. One patient developed "possibly related" sepsis one day after the third dosing but no pathogen was identified in blood cultures, and the patient recovered after a course of antibiotics. Another patient developed C. difficile diarrhea 24 hours post-administration. This was not causally related to MaaT013, as C. difficile testing was negative in the MaaT013 lot. Nosocomial transmission was suspected as the cause as several patients with C. difficile infection were hospitalized in the same unit during this period. Conclusions We herein report for the first time the treatment of 29 patients with steroid-dependent or SR intestinal aGvHD using a full ecosystem, standardized, pooled-donor, high-richness biotherapeutic. The overall response rate was 59% with the off-the-shelf MaaT013 product, which was shown to be safe and effective in these heavily pre-treated and immunocompromised patients, warranting further exploration of the full ecosystem microbiota restoration approach. Disclosures Malard: Astellas: Honoraria; JAZZ pharmaceutical: Honoraria; Sanofi: Honoraria; Janssen: Honoraria; Keocyt: Honoraria; Theralos/Mallinckrodt: Honoraria; Biocodex: Honoraria. Plantamura:MaaT Pharma: Current Employment. Mohty:BMS: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; GSK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Stemline: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Abstract: Introduction. AML standard intensive induction chemotherapy ("3+7" or equivalent) combined with wide spectrum antibiotics can dramatically alter the composition of the gut microbiota, leading to dysbiosis which is characterized by loss of microbial diversity. Such dysbiosis status can promote a pathological condition involving uncontrolled local immune responses, systemic inflammation and increased incidence of adverse events. The development of FMT-based drugs to restore microbial communities could offer novel therapeutic possibilities to reduce such adverse events and potentially improve outcomes in AML. We therefore conducted this single arm prospective phase I/II multicenter trial (NCT02928523) to evaluate the use of a FMT-based drug in association with AML induction treatment to restore the gut microbiota diversity. Patients and methods. A total of 62 consecutive patients aged between 24 and 69 years old with a diagnosis of de novo AML were screened in 7 French sites. At time of admission and AML diagnosis (Step 1=S1), patients' faeces were collected, rigorously screened, prepared following a standardized process, and stored at -80°C until later administration. The drug was administered as an enema after hematopoietic recovery (S2) and before consolidation chemotherapy (Conso). The primary endpoint was the recovery of at least 70% of microbiota diversity (based on the Simpson index) after drug administration and the reduction of multidrug resistant bacteria carriage. Blood and feces samples were collected at S1, S2, and around 10 days post-FMT before Conso (S3). Microbiome diversity restoration was assessed by metagenomics analysis through Illumina HiSeq shotgun sequencing. Antibiotic resistance gene carriage (ARGC, also known as resistome) was evaluated through mapping of readouts on the MEGARES database. Secondary objectives included safety and analysis of host response with assessment of blood and fecal markers by ELISA and Luminex. Results. Overall, 25 patients were actually treated with FMT, and 20 were included in the per-protocol population. Induction Chemotherapy (IC) induced a dramatic shift in microbial communities, with a significant 42.3% decrease of mean α-diversity Simpson index between S1 and S2 at species level (0.85 to 0.50; p 〈 0.001). Ten days after FMT administration (S3), the Simpson index returned to its initial baseline level (0.50 to 0.86; p 〈 0.001). In addition to variations of the diversity, we demonstrated using the Bray-Curtis dissimilarity index (BC) a profound shift in the microbial communities following IC (mean BC S1-S2: 0.76) and the restoration of the initial microbial profile after FMT (mean BC S1-S3: 0.40). Moreover, IC and associated antibiotic treatments induced a significant increase in the mean number of readouts mapped against antibioresistance genes at S2 (167546 to 371466 reads, p 〈 0.01) that reflect ARGC. Then, a significant reduction of 43% of the mean number of reads mapped was observed at S3 after FMT (211128 reads, p 〈 0.001). No serious adverse events (SAE) were observed within 30 days after FMT and all post FMT SAEs were not related to the FMT procedure. Moreover, FMT did not induce any local or systemic inflammatory reaction as measured by fecal and blood markers (fecal neopterin and IgA; plasmatic CRP, IL-6 and sCD14). Interestingly, restoration of the microbiome diversity was associated with a significant reduction of CRP and fecal neopterin levels, suggesting a potential anti-inflammatory impact of FMT. Overall, FMT was well tolerated and had an excellent safety profile. The one-year overall survival estimate in the whole cohort was 84% (4 deaths among 25, none of which were related to FMT: 2 multiple organ failures, 1 heart attack and 1 grade IV resistant GVHD). The median time to death from the second FMT was 182.5 days (113-225 days). Conclusions. This is the first prospective trial testing the safety and efficacy of FMT in AML patients receiving intensive induction chemotherapy. The trial achieved its primary endpoint and established the capacity of FMT to restore a diverse microbiome with high levels of similarity to baseline, as well as reducing ARGC and intestinal inflammation. A controlled randomized trial with repeated FMT administrations is currently planned to further evaluate the impact of FMT on clinical outcomes and long-term survival. (This trial was funded by MaaT Pharma whose product was tested in this protocol). Disclosures Mohty: MaaT Pharma: Consultancy, Honoraria. Doré:MaaT Pharma: Consultancy, Honoraria.