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Abstract
Background 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
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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.
Heat-map analysis of microbiome profiling using both metagenomics and qPCR probes of 107 bacteria of 24 HV treated with CZA or CZA + DAV132. Each column represents on HV. MGS, metagenomics; qPCR, Bio-ME 107 qPCR chip; CZA ceftazidime; D1, Prior to CZA initiation; D6 after CZA +/- DAV132 at 12 g po tid x 7 days
DAV132 prevents antibiotic-induced loss of anti-tumor response in murine germ-free cancer model. The germfree mice received FMT with fecal material at baseline (before ATB administration) or D6 of treatment from 3 different HV treated with CZA or with CZA + DAV132 enrolled in the clinical study. Pooled tumor growth of 3 experiments from the 3 different HV are represented for each graph. *** *p < 0.001, Mann-Whitney U Tests applied at sacrifice
DAV132 maintains an effective immune response during ATB treatment in mice. A. Heatmap representation of the 26 populations visualized in the UMAP with their respective normalized mean fluorescence intensity of the depicted markers. In grey the frequency of pooled population for each group and the statistical analysis were done based on the percentage per mouse. B. ImmuCellAI-mouse extrapolation of central memory CD8+ T cells. The center line indicates the median value, lower and upper hinges represent the 25th and 75th percentiles, respectively, and whiskers denote minimum and maximum. C. RNAseq Heatmap representing expression of genes differentially expressed on MCA-205 CD8+ T cells sorted n=5mice/group between CZA-DAV132 + Iso-PD-1 and CZA-DAV132 + anti-PD-1 and other groups (FDR adjusted p-values < 0.01). Rows: genes were clustered based on euclidean distance and «< complete » method. Columns: samples were ordered given group membership. *p < 0.05, * p < 0.01 Mann-Whitney U Tests. Statistics were performed on n = 10 mice
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