Antibiotics (ATB) induce intestinal dysbiosis and decrease the efficacy of immune checkpoint inhibitors (ICI).1,2 DAV132 is an orally administered colon-targeted ATB adsorbent designed to prevent ATB-induced dysbiosis.3 We investigated whether DAV132 co-administered with ATB could protect gut microbiota diversity and composition. Moreover, in murine avatar tumor model, we assessed anti-PD-1 efficacy through fecal microbiota transplantation (FMT) in germ-free (GF) or antibiotic-treated specific pathogen-free (SPF) mice.
Methods
Twenty-four human healthy volunteers (HV) were randomized to receive either ceftazidime-avibactam (CZA, 2g/0.5g q8h IV for 5 days) or CZA+DAV132 (12g PO tid for 7 days). CZA plasmatic and fecal pharmacodynamic levels were measured using HPLC-MS/MS. Microbiome was profiled with 16S and shotgun metagenomics at different timepoints. FMT in GF or ATB-treated SPF mice was performed using fecal samples from 3 HV and 2 HV respectively, in each group before (D1) or after 6 days (D6) of CZA+/-DAV132; subsequently mice were inoculated with MCA-205 tumor and treated intraperitoneally with anti-PD-1, 4 times every 3 days. Immunological population of tumor infiltrating lymphocytes were analyzed by flow cytometry.
Results
DAV132 did not impact plasmatic CZA concentrations, but significantly reduced ceftazidime concentration in feces compared to HV treated with CZA alone (p<0.001). DAV132 significantly prevented the reduction in microbiota alpha-diversity at D6 (p=0.0019) and was associated with a more rapid return to baseline microbiota composition (figure 1). Significantly more bacteria associated with better response to ICI were preserved in the DAV group compared to CZA, among which Faecalibacterium praunistzii and several Alistipes spp. FMT in GF mice transplanted with feces collected at D1 exhibited a significant anti-PD-1 activity. This anti-tumor response was inhibited in mice transplanted with D6 feces from any of the 3 CZA-treated HV. Conversely, the anti-tumor response was maintained in mice transplanted with D6 feces from any of the 3 HV treated with CZA + DAV132 (figure 2). Similar results were observed upon FMT using samples from HVs into ATB-treated SPF mice. Flow cytometry on tumor T cell infiltrates demonstrated that CZA decreased CD8+T cell infiltration and CD8+/Tregulatory ratio, compared to CZA + DAV132 treated HVs (figure 3).
Conclusions
DAV132 strongly prevented CZA-induced dysbiosis in HV without influencing plasmatic concentrations. In avatar mice FMT from HV treated with CZA+DAV132 was able to preserve anti-PD-1 cancer efficacy. These results provide rationale to launch clinical trials combining DAV132 in patients on ATB amenable to ICI.
Acknowledgements
This work was funded by Da Volterra, a French biotech company, through the sharing of fecal samples and a collaboration agreement with Pr. Routy's lab.
References
Derosa L, Routy B, Desilets A, Daillère R, Terrisse S, Kroemer G, Zitvogel L. Microbiota-centered interventions: the next breakthrough in Immuno-Oncology? Cancer Discov. 2021;11(10):2396–2412. Routy B, Le Chatelier E, Derosa L, Duong CPM, Alou MT, Daillère R, Fluckiger A, Messaoudene M, Rauber C, Roberti MP, Fidelle M, Flament C, Poirier-Colame V, Opolon P, Klein C, Iribarren K, Mondragón L, Jacquelot N, Qu B, Ferrere G, Clémenson C, Mezquita L, Masip JR, Naltet C, Brosseau S, Kaderbhai C, Richard C, Rizvi H, Levenez F, Galleron N, Quinquis B, Pons N, Ryffel B, Minard-Colin V, Gonin P, Soria JC, Deutsch E, Loriot Y, Ghiringhelli F, Zalcman G, Goldwasser F, Escudier B, Hellmann MD, Eggermont A, Raoult D, Albiges L, Kroemer G, Zitvogel L. Gut microbiome influences efficacy of PD-1-based immunotherapy against epithelial tumors. Science. 2018;359(6371):91–97. Vehreschild MJGT, Ducher A, Louie T, Cornely OA, Feger C, Dane A, Varastet M, Vitry F, de Gunzburg J, Andremont A, Mentré F, Wilcox MH. An open randomized multicenter Phase 2 trial to assess the safety of DAV132 and its efficacy to protect gut microbiota diversity in hospitalized patients treated with fluoroquinolones. J Antimicrob Chemother. 2022;77(4):1155–1165.
Ethics Approval
All animal studies were approved by the Institutional Animal Care Committee (CIPA) and carried out in compliance with the Canadian Council on Animal Care guidelines (Ethics numbers: C18029BRs).
Antibiotics (ATB) are known to induce gut microbiome dysbiosis and decrease the efficacy of immune checkpoint inhibitors (ICI).1 2 DAV132 oral capsule is a colon-targeted ATB adsorbent designed to prevent ATB-related dysbiosis.3 We performed a randomized study in healthy volunteers (HV) treated with distinct ATB alone or in combination with DAV132 to assess ATB plasma concentration and impact on the microbiome composition. Fecal microbiota transplants in avatar mice were performed to determine if DAV132 was able to maintain ICI response.
Methods
148 HV were randomized to receive ceftazidime-avibactam (CZA), piperacillin-tazobactam (PTZ) or Ceftriaxone (CRO) IV for 5 days alone or in combination with DAV132 orally 7.5 g or 12 g po tid for 7 days. Plasmatic and fecal pharmacodynamic were measured using HPLC. Microbiome was profiled with 16S, metagenomics and qPCR bacterial probe. In MCA-205 or B16 tumors models, FMT in GF or ATB-treated mice was performed using fecal samples from 12 HV, before CZA or PTZ+/-DAV132 or at D#6, subsequently mice received anti-PD-1. Tumor infiltrating lymphocytes were analyzed by flow cytometry and RNAseq.
Results
DAV132 did not impact plasmatic ATB concentrations, but especially at higher dose significantly reduced CZA and PTZ concentration in feces. In the CRO group, endogenous β-lactamase metabolized the ATB and fecal concentrations was almost undetectable. DAV132 at both doses did not lead to any severe adverse effect. In PTZ and CZA groups, DAV132 significantly protected microbiome diversity and was associated with a more rapid return to baseline composition (figure 1). Moreover, relative abundance more bacteria were preserved in the DAV132 groups compared to ATB alone groups, in particular A.muciniphila, Faecalibacterium and Ruminococcus. FMT in GF or ATB-treated mice revealed that the anti-tumor response was inhibited in mice transplanted with D#6 feces from HV on ATB alone groups while PD-1 response was maintained in mice transplanted with D#6 feces from HV treated with CZA or PTZA+DAV132 (figure 2). Flow cytometry analysis and RNAseq showed an upregulation of activated CD8+ T with unique gene signature in mice treated with CZA or PTZ + DAV132 compared to ATB alone (figure 3).
Conclusions
DAV132 was well tolerated and protected CZA or PTZ-induced dysbiosis without influencing ATB plasmatic concentration. In avatar mice, we showed that HV feces on ATB+DAV132 maintained anti-PD-1 response in a CD8 T cell dependent mechanism. These results provide rationale to launch clinical trials combining DAV132 in cancer patients on ATB amenable to ICI.
Acknowledgements
This work was funded by Da Volterra, a French biotech company, through the sharing of fecal samples and a collaboration agreement with Pr. Routy's lab.
Trial Registration
Clinical trial CL-006 a randomized, open-label, parallel groups, controlled study assessing the effect of DAV132 capsule filed as medical device and not as a drug to the EUDAMED with the objective to determine the plasma concentration of three ATB (CZA, PTZ and CRO) alone or in combination with DAV132 at 2 different doses (7.5 g pot id or 12 g pot id x 7 days). The study was conducted in a phase 1 centre in France according to Medical Device regulation and registered by Competent Authority (ANSM) under number: 2019-A00240-57. All investigations were conducted by investigators after HV had provided informed consent and all activities were performed according to Good Clinical Practice (GCP).
References
Routy B, Le Chatelier E, Derosa L, Duong CPM, Alou MT, Daillère R, Fluckiger A, Messaoudene M, Rauber C, Roberti MP, Fidelle M, Flament C, Poirier-Colame V, Opolon P, Klein C, Iribarren K, Mondragón L, Jacquelot N, Qu B, Ferrere G, Clémenson C, Mezquita L, Masip JR, Naltet C, Brosseau S, Kaderbhai C, Richard C, Rizvi H, Levenez F, Galleron N, Quinquis B, Pons N, Ryffel B, Minard-Colin V, Gonin P, Soria JC, Deutsch E, Loriot Y, Ghiringhelli F, Zalcman G, Goldwasser F, Escudier B, Hellmann MD, Eggermont A, Raoult D, Albiges L, Kroemer G, Zitvogel L. Gut microbiome influences efficacy of PD-1-based immunotherapy against epithelial tumors. Science. 2018 Jan 5;359(6371):91–97. Lurienne L, Cervesi J, Duhalde L, de Gunzburg J, Andremont A, Zalcman G, Buffet R, Bandinelli PA. NSCLC Immunotherapy efficacy and antibiotic use: a systematic review and meta-analysis. J Thorac Oncol. 2020 Jul;15(7):1147–1159. Vehreschild MJGT, Ducher A, Louie T, Cornely OA, Feger C, Dane A, Varastet M, Vitry F, de Gunzburg J, Andremont A, Mentré F, Wilcox MH. An open randomized multicentre Phase 2 trial to assess the safety of DAV132 and its efficacy to protect gut microbiota diversity in hospitalized patients treated with fluoroquinolones. J Antimicrob Chemother. 2022 Mar 31;77(4):1155–1165.
Ethics Approval
Clinical trial. CL-006 a randomized, open-label, parallel groups, controlled study assessing the effect of DAV132 capsule filed as medical device and not as a drug to the EUDAMED with the objective to determine the plasma concentration of three ATB (CZA, PTZ and CRO) alone or in combination with DAV132 at 2 different doses (7.5 g pot id or 12 g pot id x 7 days). The study was conducted in a phase 1 centre in France according to Medical Device regulation and registered by Competent Authority (ANSM) under number: 2019-A00240-57. All investigations were conducted by investigators after HV had provided informed consent and all activities were performed according to Good Clinical Practice (GCP). Ethics for animal studies: All animal studies were approved by the Institutional Animal Care Committee (CIPA) and carried out in compliance with the Canadian Council on Animal Care guidelines. Number of the approval: 2023-10753, C22033BRs.
2585 Background: In recent years, the gut microbiome has increasingly emerged as influencing the response to immune checkpoint inhibitors (ICIs) and antibiotic (ABX) exposure has repeatedly been shown to impair clinical outcomes of patients suffering from different cancer types and treated with ICIs. We published in 2020 a meta-analysis confirming that ABX use hampered survival of non-small cell lung cancer (NSCLC) patients treated with ICIs. The present study aims to determine whether ABX use also reduces survival of patients receiving ICIs for other cancers. Methods: PubMed and major oncology conferences’ proceedings were systematically searched to identify studies assessing the impact of ABX on the clinical outcomes of cancer patients treated with ICIs. Studies were included when reporting data on Overall Survival (OS), Progression-Free Survival (PFS), Overall Response Rate (ORR) and Progressive Disease Rate (PD), according to ABX exposure. Pooled Hazard Ratios (HRs) for OS and PFS and Odds Ratios (ORs) for ORR and PD were calculated, as well as HRs for OS and PFS according to different cancer types and different ABX exposure time windows (TWs). Results: Overall, 94 independent cohorts were included, representing 26,174 patients suffering from various types of cancer. The pooled HRs for PFS (61 cohorts, 13,224 patients) and OS (88 cohorts, 25,480 patients) were 1.47 [95% Confidence Interval (CI) 1.31-1.66] and 1.66 [95% CI 1.50-1.83], respectively, confirming a significant harmful impact of ABX on patient’ survival, observed across all cancer types (Table). The analyses of OS and PFS based on ABX exposure TWs suggested a stronger deleterious effect of ABX when taken around ICI treatment initiation. The response to treatment among ABX users was also impaired: the pooled ORs for ORR (30 cohorts, 4,590 patients) and PD (33 cohorts, 4,972 patients) were 0.55 [95% CI 0.39-0.77] and 1.97 [95% CI 1.48-2.64], respectively. Conclusions: ABX were shown to impair the clinical outcomes of cancer patients treated with ICIs, regardless of cancer type. [Table: see text]
Abstract Background: Over the last decade, studies unraveled the cancer-immune dialogue in the setting of immune checkpoint inhibitors (ICI) and influenced by the gut microbiota. The first evidence of the key role of the microbiota in ICI modulation was observed during antibiotics (ATB) treatment, where altering the microbiota composition by ATB inhibited ICI responses. DAV132 (DAV) is an orally administered colon-targeted ATB adsorbent capsules designed to prevent ATB-induced dysbiosis. We investigated whether DAV co-administered with ATB could prevent ATB-related dysbiosis and ICI response. Methods: 72 human healthy volunteers (HV) were randomized to receive either IV ceftazidime-avibactam (CZA) or Piperacillin tazobactam (PTZ) alone or in combination with oral DAV. CZA and PTZ plasmatic and fecal pharmacodynamic levels were measured using HPLC-MS/MS. Microbiome was profiled with metagenomics at different timepoints. FMT experiments in germ-free mice were performed using fecal samples from HV from the trial, before (D1) or after 6 days (D6) of CZA or PTZ+/-DAV; subsequently mice were inoculated with MCA205 or B16 tumors and treated with anti-PD-1. Tumor infiltrating lymphocytes (TILs) were analyzed by flow cytometry. Results: DAV did not impact plasmatic CZA or PTZ concentrations, but significantly reduced ceftazidime and piperacillin concentrations in feces compared to ATB groups alone. DAV significantly prevented the reduction in microbiota alpha-diversity at D6 and was associated with a rapid return to baseline microbiota. 50 and 43 metagenomics species were preserved in the CZA+DAV vs CZA, or PTZ-DAV vs PTZ such as Faecalibacterium praunistzii, Alistipes Spp and Blautia obeum. FMT in germ-free mice using feces collected at D1 exhibited a significant anti-PD-1 activity. This anti-tumor response was inhibited in two tumors models in mice transplanted with D6 feces from patients in the CZA or PTZ alone groups. Conversely, the anti-tumor response was maintained in mice transplanted with D6 feces from HV treated with CZA+DAV or PTZ+DAV groups. Flow cytometry on TILs demonstrated that CZA decreased CD8+T cell and CD8+/Treg ratio compared to CZA+DAV. Conclusions: DAV prevented ATB-induced dysbiosis in HV treated with CZA or PTZ without influencing plasmatic concentrations. In avatar mice FMT from HV treated with CZA+DAV was able to preserve anti-PD-1 efficacy. These results provide rationale to launch clinical trials combining DAV in patients on ATB amenable to ICI. Citation Format: Meriem Messaoudene, Nathalie Saint-Lu, Frédérique Sablier-Gallis, Stéphanie Ferreira, Mayra Ponce, Clément Le Bescop, Thomas Loppinet, Tanguy Corbel, Céline Féger, Fabien Vitry, Antoine Andremont, Jean de Gunzburg, Bertrand Routy. Prevention of antibiotic-induced dysbiosis in human volunteers by DAV132 and preservation of responsiveness to anti-PD-1 demonstrated by transplantation of human feces into tumor-bearing mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5882.
Immune checkpoint inhibitors (ICIs) have considerably improved patient outcomes in various cancer types, but their efficacy remains poorly predictable among patients. The intestinal microbiome, whose balance and composition can be significantly altered by antibiotic use, has recently emerged as a factor that may modulate ICI efficacy. The objective of this systematic review and meta-analysis is to investigate the impact of antibiotics on the clinical outcomes of cancer patients treated with ICIs.PubMed and major oncology conference proceedings were systematically searched to identify all studies reporting associations between antibiotic use and at least one of the following endpoints: Overall Survival (OS), Progression-Free Survival (PFS), Objective Response Rate (ORR) and Progressive Disease (PD) Rate. Pooled Hazard Ratios (HRs) for OS and PFS, and pooled Odds Ratios (ORs) for ORR and PD were calculated. Subgroup analyses on survival outcomes were also performed to investigate the potential differential effect of antibiotics according to cancer types and antibiotic exposure time windows.107 articles reporting data for 123 independent cohorts were included, representing a total of 41,663 patients among whom 11,785 (28%) received antibiotics around ICI initiation. The pooled HRs for OS and PFS were respectively of 1.61 [95% Confidence Interval (CI) 1.48-1.76] and 1.45 [95% CI 1.32-1.60], confirming that antibiotic use was significantly associated with shorter survival. This negative association was observed consistently across all cancer types for OS and depending on the cancer type for PFS. The loss of survival was particularly strong when antibiotics were received shortly before or after ICI initiation. The pooled ORs for ORR and PD were respectively of 0.59 [95% CI 0.47-0.76] and 1.86 [95% CI 1.41-2.46], suggesting that antibiotic use was significantly associated with worse treatment-related outcomes.As it is not ethically feasible to conduct interventional, randomized, controlled trials in which antibiotics would be administered to cancer patients treated with ICIs to demonstrate their deleterious impact versus control, prospective observational studies and interventional trials involving microbiome modifiers are crucially needed to uncover the role of microbiome and improve patient outcomes. Such studies will reduce the existing publication bias by allowing analyses on more homogeneous populations, especially in terms of treatments received, which is not possible at this stage given the current state of the field. In the meantime, antibiotic prescription should be cautiously considered in cancer patients receiving ICIs.https://www.crd.york.ac.uk/prospero/, identifier CRD42019145675.
Immune checkpoint inhibitors (ICIs) have been shown to improve patients' clinical outcomes in a variety of cancers, but with variable efficacy. Prior research has also suggested that systemic antibiotic (ABX) exposure may impact the intestinal microbiota and result in suboptimal ICI treatment outcomes. Our team published a systematic review and meta-analysis showing that ABX use could indeed decrease the survival of patients diagnosed with non-small-cell lung cancer (NSCLC) and treated with ICIs.1 The present abstract aims at updating this meta-analysis by incorporating new studies that have been published in the period ranging from September 2019 to August 2020.
Methods
Medline (through PubMed), the Cochrane Library and major oncology conferences proceedings were systematically searched to identify studies assessing the impact of ABX use on the clinical outcomes of NSCLC patients treated with ICIs. Studies were found eligible for inclusion when they mentioned a hazard ratio (HR) or Kaplan–Meier curves for overall survival (OS) or progression-free survival (PFS) based on antibiotic exposure. Pooled HRs for OS and PFS and HRs for OS and PFS according to different time windows for ABX exposure were calculated.
Results
6 eligible new studies were identified between September 2019 and August 2020 while 3 other studies were updated with new information. Altogether, 27 studies reported data for OS (6,436 patients, 826 of whom coming from new studies) and 24 for PFS (3,751 patients, 786 of whom coming from new studies). The pooled HR was 1.75 (95% confidence interval [CI]: 1.38–2.23) for OS and 1.57 (95% CI: 1.28–1.92) for PFS, confirming a significantly reduced survival in patients with NSCLC exposed to ABX. The detailed analysis in subgroups based on the time window of exposure (figure 1, figure 2) suggests that the deleterious effect of ABX is stronger when the exposition happens shortly before and after the initiation of the ICI treatment.
Conclusions
The update of the meta-analysis confirms the previously reported deleterious effect of ABX on ICI treatment outcomes, taking into account the latest publications in the field. The topic deserves further research to uncover if the effect will stand with 1st line use of ICI together with chemotherapies and/or other approved combinations, elucidate the mechanisms at stake and improve care of patients.
Reference
Lurienne L, Cervesi J, Duhalde L, de Gunzburg J, Andremont A, Zalcman G, et al. NSCLC immunotherapy efficacy and antibiotic use: a systematic review and meta-analysis. J Thorac Oncol 2020;15:1147–1159.
