key: cord-0959616-hastxbu2
authors: Daillère, Romain; Derosa, Lisa; Bonvalet, Mélodie; Segata, Nicola; Routy, Bertrand; Gariboldi, Manuela; Budinská, Eva; De Vries, I. Jolanda M.; Naccarati, Alessio Gordon; Zitvogel, Valérie; Caldas, Carlos; Engstrand, Lars; Loilbl, Sibylle; Fieschi, Jacques; Heinzerling, Lucie; Kroemer, Guido; Zitvogel, Laurence
title: Trial watch : the gut microbiota as a tool to boost the clinical efficacy of anticancer immunotherapy
date: 2020-06-03
journal: Oncoimmunology
DOI: 10.1080/2162402x.2020.1774298
sha: 6df83852e19b5fd3d9bce888b60eeb2944e524a4
doc_id: 959616
cord_uid: hastxbu2

Accumulating evidence demonstrates the decisive role of the gut microbiota in determining the effectiveness of anticancer therapeutics such as immunogenic chemotherapy or immune checkpoint blockade in preclinical tumor models, as well as in cancer patients. In synthesis, it appears that a normal intestinal microbiota supports therapeutic anticancer responses, while a dysbiotic microbiota that lacks immunostimulatory bacteria or contains overabundant immunosuppressive species causes treatment failure. These findings have led to the design of clinical trials that evaluate the capacity of modulation of the gut microbiota to synergize with treatment and hence limit tumor progression. Along the lines of this Trial Watch, we discuss the rationale for harnessing the gut microbiome in support of cancer therapy and the progress of recent clinical trials testing this new therapeutic paradigm in cancer patients.

The rise and success of cancer immunotherapy over the past decade has revolutionized the clinical management of a wide array of malignancies that were previously associated with poor prognosis. 1 At the forefront of immunotherapy development are immune-checkpoint blockers (ICBs), which have seen enormous and unparalleled success in cancer therapy as a result of their broad bioactivity across many histological tumor types, the durability of their responses, and therapeutic success stories that sometimes involve even metastatic and chemo-resistant diseases. [2] [3] [4] [5] [6] [7] [8] Antibodies targeting the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) or the interaction between programmed cell death protein 1 and programmed cell death ligand 1 (PD-1/ PD-L1) disrupt negative immune regulatory checkpoints, thus unleashing antitumour immune responses. Such ICBs have been approved by the regulatory agencies and are now being considered as standard of care in a wide range of solid and hematologic neoplastic diseases including advanced-stage melanoma, non-small-cell lung cancer (NSCLC), head and neck cancer, bladder or renal cell cancer (RCC). 9 In spite of the exceptional improvement in objective response rates and overall survival in a minority (~30%) of patients, ICBs responses are heterogeneous (with occasional acceleration of the disease called "hyperprogression" and a majority of patients exhibiting primary resistance) and sometimes transient (meaning that therapeutic "success" is followed by secondary resistance). Large efforts are being dedicated to identify the parameters that govern the strength, timing and the threshold of the anticancer immunity needed to trigger the effectiveness of anticancer therapeutics, a notion defined as the "cancer immune set-point". This setpoint would dictate the capacity of a particular cancer patient to mount an effective antitumor immunity counteracting tumor progression. 10, 11 Among the factors deciphering the mechanisms of primary resistance to ICBs, evidence accumulating over the past decade has highlighted the role of the gut microbiota. Indeed, the reciprocal relationship between cancer progression, immune control, primary resistance/sensitivity to ICBs and the microbiota is becoming increasingly apparent. [12] [13] [14] [15] The role of the gut microbiome in modulating the cancer-immune set-point

The human gut microbiome modulates many host processes, including metabolism, inflammation, immune and intestinal epithelial cell responses. [16] [17] [18] In the last decade, major progress has been made in the comprehension of cancer development in interaction with the microbiota. 19 Indeed, a 'deviated' repertoire of the gut microbiome, that has been referred to as 'intestinal dysbiosis' has been epidemiologically -and sometimes causally -associated with a variety of chronic inflammatory disorders. In parallel, discoveries made in preclinical tumor models and in cancer patients have demonstrated that the composition of the intestinal microbiota influences the effectiveness of anticancer drugs (such as immunogenic chemotherapies and ICBs) and regulates tumor immunosurveillance. [20] [21] [22] [23] [24] [25] [26] [27] Several lines of evidence have unraveled the link between the gut microbiota and ICBs-mediated anti-tumor immune responses. Hence, studies performed in axenic (gnotobiotic) or broad-spectrum antibiotic-treated mice have supported a cause-effect relationship between dysbiosis and the failure of anticancer therapeutics. [20] [21] [22] 25, 27 Similar retrospective and prospective studies in advanced cancer patients across a diverse range of malignancies and geographic locations revealed that antibiotic treatment before anticancer therapeutics dampens the clinical efficacy of ICBs and immunogenic chemotherapy, highlighting that the disruption of a homeostatic microbiome (i.e. a switch from eubiosis to dysbiosis) and the loss of specific bacterial species may be detrimental for the success of anticancer therapies. [27] [28] [29] [30] [31] [32] [33] [34] In line with this notion, Derosa et al. European Urology (In press, DOI : 10.1016/j.eururo.2020.04.044) showed that antibiotics prior to immune checkpoint inhibitors had a deleterious clinical impact, reduce the micobiome diversity and increase Clostridium hathewayi bacteria associated with resistance.

In addition, the functional properties of the intestinal microbiota can be studied by transferring the entire fecal microbiota from patients to axenic or antibiotic-pretreated recipient mice, a process that is called fecal microbial transplantation (FMT). Experiments in mice have established robust cause-effect relationships between the composition of the gut microbiota and the therapeutic outcome of immune-based anticancer therapeutics. Indeed, the phenotype of patients (that are either clinical responders or non-responders to ICBs) can be recapitulated in mice through FMT, demonstrating that the fecal material derived from cancer patient drives the capacity to respond to ICBs. In line with these observations, such mice can be considered to serve as 'avatars' of the patientderived microbiota. 22, 24, 26, 27 Recent advances in sequencing methods studying the composition of the intestinal microbiota have ameliorated our capacity to unravel correlations between specific gut microbiota fingerprints and the onset or course of pathological processes. 35 Accordingly, exploration of the composition of the gut microbiota in cancer patients through 16 S rRNA sequencing or quantitative metagenomics has demonstrated a major impact of the gut microbiota on the clinical activity of immune checkpoint inhibitors. Indeed, a triad of papers published in 2018 in Science support the notion that the composition of the gut microbiota modulates the response to immunotherapy with PD-1 or PD-L1 blocking antibodies, including non-small cell lung cancer, renal cell carcinoma and melanoma. 24, 26, 27 In all three studies, independent patient populations were subjected to fecal microbial analyses, leading to the identification of bacterial entities that positively correlate with the clinical outcome according to the response evaluation criteria in solid tumors criteria (RECIST). This methodology has led to the identification of several bacterial species that favor anticancer immunosurveillance. Accordingly, transfer of defined bacterial species is capable of restoring responses in a variety of preclinical tumor models. For example, supplementation with Akkermansia muciniphila or Enterococcus hirae, two species that are associated with a favorable clinical outcome of PD1 blockade in NSCLC patients, reestablish the capacity of the murine immune system to mediate ICBsstimulated anticancer responses in tumor-bearing avatar mice. 27 Additional studies have demonstrated the capacity of several other bacterial species such as Bacteroides fragilis, Bifidobacterium longum, Barnesiella intestinihomins or Alistipes shahii to support anticancer immunity by activating dendritic cells (DCs), by stimulating the production of interleukin-12 (IL-12) by DCs, by enhancing recruitment of tumorspecific cytotoxic T lymphocytes (CTLs), by triggering the production of interferon-γ (IFNγ) by tumor-infiltrating γδ T cells or by elevating the production of TNFα by intratumoural myeloid cells. [21] [22] [23] 25 Importantly, memory T cell responses associated with IFNγ production by peripheral blood CD4 + and CD8 + T cells upon co-culture with A. muciniphila or E. hirae-pulsed peripheral blood autologous monocytes were stronger in patients with NSCLC or RCC who responded to ICBs than in nonresponders and predicted longer progression-free survival (PFS) in these cohorts of patients. 21, 27 Interestingly, such an immune reactivity against E. hirae or B. longum also correlates with robust CD8 + T cell responses and better prognosis in HBV-related hepatocellular carcinoma, 36 potentially highlighting the clinical relevance of these particular strains across different malignancies.

Therefore, accumulating evidence brings to light that modulating the composition of the gut microbiota and harnessing the immunogenicity of the intestinal microbiome may become promising strategies to circumvent primary resistance to anticancer therapeutics ( Figure 1 ).

William Coley has been the first to develop a combination of microbial products including Streptococcus pyogenes and Serratia marcescens which has proven antitumoral efficacy. 37 Following this pioneering discovery, multiple attempts have been launched to use microbial agents for anticancer therapy. Since 1990, Mycobacterium bovis Bacille Calmette Guérin (BCG) has been approved by both FDA-and EMA for the treatment of noninvasive bladder cancer. 38 BCG induces a local immune response against residual cancer cells that largely reduces the probability of relapse. 39, 40 No other bacteria have reached clinical approval by the regulatory agencies so far.

Although the development of anticancer agents based on live microbial agents has traditionally focused on parenteral (local or systemic) administration, investigators have recently been considering to orally administer live bacteria to safely boost the clinical efficacy of anticancer treatments or diminish toxicities associated to these therapeutics. As such, interventional approaches aiming at modulating the composition of the gut microbiota are under development and include fecal microbiota transplantation (FMT), antibiotic regimens, prebiotic and/or probiotic formulations, or other types of drug (such as the diabetes drug metformin) and dietary-based interventions, such as caloric restriction. 41 To date, multiple clinical trials (www.ClinicalTrials.gov) are investigating the antitumoral potential of live biotherapeutic products (LBPs), ie FMT, single strain bacteria or bacterial consortia ( Table 1 ). The majority of these clinical trials focus on cancer patients that previously failed immunotherapy. As such, the capacity of microbial products to convert nonresponders cancer patients into responders is under evaluation (NCT03341143/NCT03353402/NCT04116775/NCT04130763/ NCT04264975/NCT03637803/NCT03595683/NCT03775850). In parallel, strategies aiming at boosting the objective response rate to ICBs in ICBs-naïve patients are investigated (NCT04056026/NCT03686202/NCT03595683/NCT03817125/ NCT04208958). Several clinical trials are testing, in the neoadjuvant setting, the capacity of LBPs to modulate the tumor microenvironment before tumor resection (NCT04139993/ NCT03934827/NCT04193904). Other clinical trials are exploring the capacity of FMT to mitigate ICBs induced-diarrhea or colitis in cancer patients, therefore allowing to uncouple efficacy from toxicity (NCT04038619/NCT04040712/ NCT04163289/NCT03819296). Last but not least, the capacity of the gut microbiota to circumvent corticosteroid-resistant acute graft-versus-host disease (GvHD) in hematologic malignancies is under investigation (NCT03812705/NCT02928523/ NCT03678493/NCT03359980). Safety, engraftment of the microbial product, monitoring of the immune compartments as well improved objective response rates and survival are generally the main endpoints of such trials. The website ClinicalTrials.gov informs on multiple clinical trials that are either ongoing or completed, yet generally lack published information on the outcome ( Table 1 )

FMT has been the first strategy to enter clinical evaluation. FMT involves the transfer of lyophilized and encapsulated feces from donor, orally or rectally. This intervention has been demonstrated to be effective in other therapeutic areas such as the management of refractory Clostridium difficile infection. 42 Two clinical trials (NCT03341143 and NCT03353402) investigate the capacity of the gut microbiota to rescue the clinical efficacy of ICBs in metastatic melanoma patients who failed previously immunotherapy. Patients with metastatic melanoma who achieved a durable complete response to treatment are serving as FMT donors. The first Phase II clinical trial (NCT03341143) has enrolled 12 patients in which 8 patients were evaluable. Among them, one patient exhibited a complete response (CR), another one a partial response (PR), two patients stable disease (SD) while the therapeutic intervention was ineffective in the remaining four cancer patients Figure 1 . Therapeutic strategy involving microbial products to circumvent primary resistance to anticancer treatments. Preliminary data suggest the capacity of gut onco-microbiome signatures to predict the clinical outcome of anticancer treatments. Data from these analyses can inform on the microbiota in cancer patients and indicate which bacterial genera or species could be beneficial to patients. The identification, cultivation and functional characterization of cancer-relevant microbial species through an extensive preclinical validation process will be crucial for optimizing the process. The development of diagnostic tools predicting the response of a particular cancer patient to a particular microbial product will allow to personalize therapeutic interventions, that will rely on fecal microbial transplantation (FMT), consortia of bacterial strains or single-strain bacteria. The outcome of microbial interventions can be evaluated by monitoring the clinical efficacy of anticancer treatments as well as the incidence of immune-related adverse effects (irAEs) or signs of graft-versus-host disease (GvHD). (https://www.umms.org/sjmc/-/media/files/um-sjmc/health-ser vices/cancer-institute/symposium-powerpoints/role-of-commen sal-microdata-in-carcinogenesis-and-cancer-therapy. p d f ? u p d = 2 0 1 9 1 1 2 0 2 1 4 4 4 5 & l a = e n & h a s h = EA5670957E87F12C6434A047801E4E0C632910B6). In the second Phase I clinical trial (NCT03353402), three out of five patients had a partial or complete response to treatment post-FMT. Interestingly, immunohistochemical staining of biopsies demonstrated an infiltration of the melanoma by antigen presenting cells (CD68 + ) as well as intra-tumoral CD8 + T-cell post-FMT. 43 Although too preliminary to draw any definitive conclusions, these two trials represent the first clinical evidence that the gut microbiota may have an impact on antitumor immunity and potentially even responses to ICBs. Another strategy consists in providing lyophilized and encapsulated single strain bacteria for oral delivery to cancer patients. A Phase I/II clinical trial (NCT03637803) investigates the safety and efficacy of MRx0518, a bacterial strain of Enterococcus gallinarum, in combination with KEYTRUDA® in cancer patients with solid tumors and advanced malignancies who have progressed on PD-1/PD-L1 inhibitors. Two out of six cancer patients displayed a partial response with evidence of increased tumor infiltrating lymphocytes (https://www.london stockexchange.com/exchange/news/market-news/market-newsdetail/DDDD/14295955.html). In contrast, a clinical trial (NCT03775850) investigating the capacity of Bifidobacterium longum (EDP1503) to boost the efficacy of pembrolizumab in microsatellite stable colorectal cancer who had previously failed all therapies did not observe any formal clinical responses, atlhough some patients manifested extended stable disease (https://evelobio.com/portfolio/).

It is also well documented that GvHD following hematopoietic stem cell transplantation has a high mortality rate. Therapeutic options such as steroids are not always efficient, indicating that GvHD remains an unmet medical need. The role of the gut microbiota in the development of GvHD has been extensively elucidated. [44] [45] [46] The first pilot clinical study (NCT02733744) exploring the administration of oral FMT capsules to patients early after allo-HCT has unraveled the feasibility, safety and efficiency of this procedure. 47 FMT was associated with an increase in recipient microbiome diversity, while the Kaplan-Meier estimates for 12-month OS post-FMT increased to 85%. 47 An additional study (NCT03359980) has released preliminary data demonstrating that 3 out 8 steroidrefractory intestinal acute GvHD attained a complete response post-FMT (external presentation, ASH2019).

ICBs can induce immune-related adverse effects (irAEs) in some cancer patients such as colitis. 48, 49 Two patients experiencing refractory ICI-associated colitis were treated with FMT from healthy donors. They exhibited a complete resolution of colitis-associated clinical symptoms, demonstrating that the gut microbiome is a potent tool for improving immune checkpoint inhibitors-associated irAEs. 50

Preliminary data suggest that the gut microbiota can safely boost the clinical efficacy of anticancer therapeutics and favor the success of hematopoietic stem cell transplantation (HSCT) in cancer patients ( Figure 1) . Interestingly, FMT-based clinical trials revealed that the efficacy of transplantion is donordependent, demonstrating the clinical relevance of specific gut microbiota signatures and raising the question as to how identify optimal donors. Several investigators are recruiting FMT donors among cancer patients who previously responded to their treatments while others are preferring healthy volunteers. The identification and functional characterization of the key bacterial species driving the favorable clinical outcome of ICBs seem crucial. In addition, FDA has issued safety alerts after the death of patients receiving FMT for Clostridium difficile infection that developed infections caused by enteropathogenic bacteria contained in the FMT. Besides, the current SARS-CoV-2 pandemics instructs us that harmful viruses for humans that have not only a tropism for the lung tissues but also for the intestinal epithelium might jeopardize the long term future of these "allogeneic FMT" based-approaches. Screening for covid in FMT trial will become mandatory. Furthermore, in one case, the obese phenotype has been transferred from a donor to a recipient, 51 calling for guidelines to exclude donors with any kind of pathology (including obesity) from the clinical protocols. Beyond obvious hygiene-related practicalities, challenges to FMT include the selection of optimal donors and the provision of sufficient material to enable long-term, repeated treatment of multiple patients. 52 Beyond these headlines, whether the donor FMT should exhibit short (within the first 3 months of ICBs) or long term (>18 months) persistance and "colonize" the recipient intestines remains an open conundrum in oncology. Finally, the necessity of a concomitant nutritional intervention or a prebiotic usage in conjunction with FMT will have to be evaluated in second generation trials.

Clinical trials will presumably establish the therapeutic impact of the gut microbiota in this scenario. However, it appears that only a subset of patients benefits from these innovative anticancer therapies, either because the recipient exhibits a primary resistance to ICBs independent from intestinal dysbiosis or because the donor FMT has not provided the appropriate set of microbes to this recipient host. Hence, there is an urgent need for suitable, robust and reliable diagnostics tools to fully identify and functionally characterize the minimalist commensal ecosystems relevant to cancer, in order to prospectively validate cancer-associated gut microbiome fingerprints of high clinical relevance. Several european (such as ONCOBIOME: https://www.oncobiome.eu/) and international consortia are currently developing "Gut Oncomicrobiome Signatures" (GOMS) across various malignancies and geographical locations (and other confounding factors), that will eventually become part of the oncological arsenal for the optimization and personalization of therapy in the future. Another issue is the capacity to manufacture microbial products at an industrial scale and with consistent levels of quality. Last but not least, it is unlikely that "one size will fit all cancer types and anticancer therapeutics". Preclinical studies that precisely define the mechanisms of action of microbial products are therefore crucial to adapt the use of microbial products to cancer patients.

As such, the gut microbiota appears to dictate the clinical efficacy of anticancer therapeutics. Without doubt, the intestinal microbiota is one of the parameters modulating the cancer immune set-point and whose therapeutic manipulation has to be incorported into the oncological arsenal. Multiple clinical trials are on the verge to be launched in this moving area. We expect the confirmation that FMT constitutes a viable and safe procedure on cancer during this calendar year. Moreover, it can be anticipated that technologies to expand the donor microbiota in bioreactors will be developed to standardize the process and provide unlimited amounts of material for FMT. As an alternative, microbially defined bacterial communities (oligoclonal consortia) or single bacterial strains (monoclonal therapies) will be developed for prolonged therapeutic interventions in cancer patients (Figure 1 ).

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RD is a full-time employee of everImmune, a biotech company dedicated to immunostimulatory bacteria. RD, GK and LZ are the scientific cofounders of everImmune. BR is on the scientific board of Vedanta.