Harnessing the Gut Microbiota to Potentiate the Efficacy of CAR T Cell Therapy

This is the authors' abstract. We don't add key points for this paper.

Chimeric antigen receptor T cell (CART) therapy has shown outstanding results in hematological malignancies.1–13 To date, 6 engineered CART products have been approved by US Food and Drug Administration and European Medicines Agency - European Union for B cell non-Hodgkin lymphoma (NHL), B cell acute lymphoblastic leukemia (B-ALL), and multiple myeloma.2–13 Despite the high rate of responses, disease refractoriness or relapse are common, and therefore, there is a growing interest in understanding the determinants of CART immunotherapy outcomes.14–17 Several mechanisms of CART resistance have been identified, including product-related factors (ie, reduced CAR T cell persistence and T cell exhaustion) and tumor-intrinsic factors (ie, antigen-negative escape, overexpression of immunomodulatory molecules, impairment of apoptotic pathways, and the complex anti-inflammatory tumor microenvironment composition).17 Various strategies have been proposed to address these issues,18,19 but a deeper knowledge of additional and concomitant causes of failure is direly needed. In fact, for many patients, it is still not possible to precisely pinpoint the cause of disease relapse or progression. In the last 2 decades, the role of gut microbiota has been deeply investigated in several human diseases, including autoimmunity, neurodegeneration, metabolic and cardiovascular diseases, cancer, and others.20–23 In particular, the gut microbiota has been recognized as a critical modulator of immune response after immune checkpoint blockade (ICB)24–27 and in allogeneic hematopoietic stem cell transplantation (allo-HCT).28,29 Initial clinical evidence suggests that the gut microbiota may also influence the antitumor effects of CAR T cell therapy.30–33 In this perspective article, we review the currently available evidence of the role of the microbiota in CART, including preclinical and clinical published data. Despite the limited current clinical evidence, we attempt to speculate on the potential therapeutic approaches that could be developed to modulate the microbiota and enhance CART efficacy and reduce toxicity. The human gut microbiota is a complex microcosm of commensal, symbiotic, or pathogenic microorganisms, including bacteria, viruses, fungi, and archaea, that modulate inflammatory, metabolic, immunological, and kinetic processes in the host. While the gut microbiota remains the most commonly studied, the microbiota of the skin, oral cavity, respiratory system, vagina, and tumors are increasingly recognized for their associations with human diseases and therapeutic outcomes.34 Since the gut microbiota was recognized as an essential regulator of the immune system, several groups have investigated its role in cancer, highlighting a close relationship between gut microbiota disruption and tumorigenesis, therapeutic effects, tumor escape, and modulation of immune responses.35 Early evidence of its role in immunotherapy came from studies on ICB, such as anti-CTLA-424,36,37 and anti-PD-1 therapies.25–27Bifidobacterium and Akkermansia muciniphila were associated with therapeutic efficacy, as they enhanced dendritic cell activity and CD8+ T cell response in the tumor microenvironment.26,27,38,39 These bacteria have been implicated in antitumor responses, and autoimmune diseases and tolerance, revealing the extensive involvement of the gut microbiota in systemic immune activity.40 Besides ICB, the role of the gut microbiota has been clinically investigated in the setting of chemotherapy, radiation therapy, and allogeneic stem cell transplant. Yoon et al have recently reported that gut dysbiosis is already present at diagnosis in a cohort of treatment-naïve, newly diagnosed patients with diffuse large B-cell lymphoma, and a high abundance of Enterobacteriaceae during R-CHOP chemo-immunotherapy (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone) correlates with febrile neutropenia and poor survival.41 In comparison, Lachnospiraceae and Enterococcaceae have been linked to postradiation restoration of hematopoiesis and gastrointestinal repair, revealing a possible protective role against gastrointestinal dysfunction in leukemia patients undergoing radiotherapy.42 Moreover, the effects of high-dose chemotherapy on fecal diversity have been studied in a cohort of autologous-HCT recipients, in which a low gut microbiota diversity has been associated with decreased progression-free survival.43 In the setting of allo-HCT, myeloablative conditioning regimens with chemo and high-dose radiation have been shown to dramatically reduce the alpha diversity of the gut microbiota,44 a measure of the unique bacteria present and their relative frequencies, which is significantly correlated with post-allo-HCT survival and disease relapse.28,29,45 Analyses of clinical data have demonstrated a strong relationship between the abundance of Blautia46 and Clostridia47 and acute graft-versus-host disease (GVHD). Furthermore, a mechanistic link between Enterococcus domination and GVHD has been established.48 Conversely, the presence of Lachnospiraceae has been associated with a reduced occurrence of chronic GVHD.49 In addition, the obligate anaerobes Faecalibacterium, Ruminococcus, and Akkermansia were strongly associated with immune reconstitution after allo-HCT, whereas Staphylococcus abundance affected the lymphopoiesis.50,51 The gut microbiota exerts significant control over the expression of major histocompatibility complex (MHC) class II on intestinal epithelial cells, thereby influencing the promotion of GVHD.52 Notably, the administration of oral vancomycin peritransplantation has been shown to reduce bacterial MHC II inducers, leading to a decrease in CD4+ T cell-mediated GVHD. This result has been recently corroborated in a large clinical allo-HCT cohort.53 Overall, these data demonstrate that the microbiota is fundamental for immune regulation and influences the outcomes of anticancer therapies. Three clinical studies on the role of the microbiota in CART have been published so far. Despite the retrospective nature of the analyses, these preliminary data suggest that the gut microbiota influences the response and toxicity to CART immunotherapy (results summarized in Table 1). In the first of these studies, conducted by Smith et al, a large cohort of patients with relapsed or refractory B-ALL and large B-cell lymphoma received anti-CD19 CART at 2 institutions, the Memorial Sloan Kettering Cancer Center and the University of Pennsylvania, using both CD28 and 4-1BB costimulated CART products.30 As expected, in 228 patients with NHL and B-ALL, exposure to antibiotics in general and in particular to broad-spectrum anaerobe-targeting ones (piperacillin-tazobactam, imipenem-cilastatin, and/or meropenem [PIM]) within 4 weeks before CART administration corresponded with decreased alpha diversity. More importantly, preinfusion PIM exposure was associated with reduced progression-free survival and overall survival in a multivariable regression model after adjusting for potential confounders. In addition, within the lymphoma subcohort, individuals exposed to antibiotics before CART infusion exhibited a higher incidence of immune effector cell-associated neurotoxicity syndrome. Conversely, in a separate prospective cohort of 48 patients, high abundance of obligate anaerobes such as Ruminococcus, Bacteroides, Faecalibacterium, and Akkermansia was associated with complete response at day 100.30 These results were consistent with the observations derived from previous studies of ICB, where a favorable gut microbiota (high diversity and abundance of Ruminococcaceae and Faecalibacterium) promoted antigen presentation and improved effector T cell function in patients treated with anti-PD-1 therapy.25–27 More recently, Stein-Thoeringer et al confirmed these data in a cohort of American and German patients affected by NHL who received axi-cel, tisa-cel, or liso-cel infusion but were not exposed to antibiotics in the 3 weeks before CART infusion.33Bifidobacterium longum abundance and peptidoglycan biosynthesis strongly correlated with long-term survival and response to CART, independently of demographic or clinical variables. Furthermore, Lachnospira pectinoschiza and Akkermansia muciniphila significantly correlated with CD3+ and CD4+ T cell counts at the time of T cell apheresis, while Bacteroides, Blautia, and Faecalibacterium prausnitzii negatively impacted on CD3+ and CD8+ T cell levels.33 Finally, the authors highlighted Akkermansia as potentially involved in better quality and performance of the manufactured T cell product. In line with the above-mentioned studies, Hu et al described a different pattern of intestinal microbes in multiple myeloma patients who achieved complete remission (CR) after anti-BCMA CART treatment31 (Table 1). Noteworthy, they found different amino acid metabolism pathways enriched in responders and nonresponders and observed that Bifidobacterium was enriched in CR patients and associated with cytokine release syndrome (CRS). In contrast, they were unable to analyze the microbiome in relation to neurotoxicity incidence and severity due to the small number of events in the cohort. Whether the microbiota influences CRS remains an open question, because neither Smith et al nor Stein-Thoeringer et al reported a correlation between gut microbiota composition and CRS. In particular, it is important to determine which antibiotics are more toxic to the gut microbiota and consequently reduce CART efficacy and increase toxicity. For example, in the retrospective cohort by Smith et al, the authors compared 2 commonly used antibiotics for the treatment of neutropenic fever: piperacillin-tazobactam and cefepime. Interestingly, piperacillin-tazobactam, a known anaerobe-targeting antibiotic that importantly affects the microbiota composition, was associated with reduced progression-free survival and overall survival as compared with cefepime. While a randomized trial would be needed to confirm a differential effect of the 2 antibiotics, it is becoming clear that the use of specific antibiotics might impair CART function. Lastly, it is fundamental to study the impact of antibiotics also in the days and weeks after CART infusion, when their administration is tightly linked to CRS and frequently requires broad-spectrum antibiotic therapy due to the challenging differential diagnosis with sepsis. Pathogen-directed treatments, time-reduced antibiotic exposures, or even only observation in nonneutropenic patients could minimize the dysbiosis and the perturbation of the immune system. In addition, the incidence of CRS is directly related to high tumor burden,1,4,10,12 and therefore its detrimental effect may be reduced by tailoring therapies to achieve better response before CART infusion. Table 1 - Gut Microbiota Composition and Outcomes in CART-treated Patients Publication Disease Patients, n (%) CART, n (%) AB Exposure, n (%) Response Toxicity CR at 3 mo, n (%) PFS(non-AB vs AB) OS(non-AB vs AB) CRS (non-AB vs AB) ICANS (non-AB vs AB) Exposure to AB pre-CART infusion (Smith et al., 2022) NHLALL 137 (60.1)91 (39.9) Anti-CD19, 228 (100):Axi-cel, 72 (31.6) Tisa-cel, 101 (44.3)19–28z, 55 (24.1) No AB, 83 (36.4)Any AB, 145 (63.6) Yes, 117 (51.3)No, 111 (48.7) NHL-ALL: HR 1.71 (1.12–2.59; P = 0.011) NHL: HR 1.29 (0.82–2.01; P = 0.256) NHL: HR 2.54 (1.41–4.56; P = 0.001) P = 0.179 P = 0.013 P-I-M, 47 (20.6) NHL-ALL: HR 2.58 (1.68–3.98; P < 0.001) P = 0.058 P = 0.023 NHL: HR 1.83 (1.03–3.27; P = 0.038) NHL: HR 3.37 (1.77–6.44; P < 0.001) P = 0.154 P = 0.002 ALL: HR 1.96 (1.15–3.35; P = 0.012) ALL: HR 2.12 (1.2–3.76; P = 0.008) P = 0.525 P = 0.254 Microbiota analysis:NHLALL 48 (100)46 (95.8)2 (4.2) 19–28z, 2 (4.2)Axi-cel, 21 (43.8) Tisa-cel, 23 (47.9)Brexu-cel, 2 (4.2) Reduced alpha diversity in CART-treated patients (P = 0.0023).Reduced beta diversity in CART-treated patients (P < 0.001). Yes, 23 (47.9)No, 25 (52.1) High abundance of Ruminococcus, Bacteroides, Faecalibacterium in CR; Akkermansia the top enriched dominant taxon in responders;High abundance of Veillonellales, family Veillonellaceae in NR. Blautia, Ruminococcus, Bacteroides, Faecalibacterium associated with the absence of toxicities. Microbiota modifications pre- and post-CART in MM (Hu et al., 2022) MM 99 (100) ORR 91 (95)aCR 53 (55.8)a Microbiota analysis:MM 81 (81.8) Anti-BCMA, 81 (100) Higher alpha diversity in CR vs PR/NR patients; Bifidobacterium, Prevotella, Colllinsella, Sutterella enriched in patients in CR pre- and post-CART; Sutterella associated with prolonged PFS post-CART. High abundance of Bifidobacterium and Leuconostoc in patients with CRS. Exposure to ABT pre-CART infusion(Stein-Thoeringer et al., 2023) NHL 172 (100) Anti-CD19:Axi-cel, 122 (70.9)Tisa-cel, 49 (28.5)Liso-cel, 1 (0.6) No AB, 110 (64)Any AB, 62 (36) Yes, 82 (47.7) HR 2.04 (1.38–3.00; P = 0.0009) HR 2.39 (1.46–3.91; P = 0.0027) High-risk ABb, 36 (20.9) No, 90 (52.3) HR 3.05 (1.96–4.75; P < 0.0001) HR 2.99 (1.69–5.29; P = 0.0003) P = 0.085 43.4% vs 63.8% (P = 0.039) Microbiota analysis: NHL 116 (67.4) Reduced alpha diversity in high-risk AB-treated patients;High abundance of Prevotella, Veillonella, Enterococcus spp. in high-risk AB-treated patients. Pre-CART high abundance of Bacteroides, Ruminococcus, Eubacterium and Akkermansia in CR patients; Bifidobacterium longum associated with response at 6 mo post-CART;High Bacteroides stercoris in NR patients. aResponse at 3 months was assessed in the efficacy analysis cohort of 95 patients.bHigh-risk AB: piperacillin/tazobactam, meropenem, cefepime, ceftazidime.AB = antibiotics; ALL = B-cell acute lymphoblastic leukemia; Axi-cel = axicabtagene ciloleucel; Brexu-cel = brexucabtagene autoleucel; CART = chimeric antigen receptor T cell therapy; CR = complete response; CRS = cytokine releasing syndrome; HR = hazard ratio; ICANS = Immune effector cell-associated neurotoxicity syndrome; NHL = non-Hodgkin lymphoma; NR = no response; ORR = overall response rate; OS = overall survival; PFS = progression-free survival; P-I-M = piperacillin/tazobactam, imipenem, meropenem; PR = partial response; Tisa-cel = tisagenlecleucel. Antibiotic-induced dysbiosis and its effects on immunotherapy outcomes have also been studied in preclinical models. The first observation on CART came from Kuczma et al, who reported that the administration of broad-spectrum antibiotics did not dampen the tumor killing of anti-CD19 CART in murine models of A20 B-cell lymphoma receiving pre-CART lymphodepletion with cyclophosphamide (CTX). Of note, the alteration of the gut microbiome was significantly associated with prolonged persistence of CART and duration of B-cell aplasia in these mice.54 Interestingly, the antitumor effect of CTX was reduced by antibiotics in the CTX-only treated mice, confirming the dependence of CTX efficacy on its immunomodulatory effects.55 In contrast, Uribe-Herranz et al recently demonstrated that immunocompetent mice receiving oral vancomycin—a nonabsorbed Gram-positive bacterium-specific antibiotic—after murine anti-CD19 CART administration experienced better control of A20 lymphoma and B16 melanoma tumors as compared with mice treated with anti-Gram-negative antimicrobials or antibiotic-naïve.32 Mechanistically, the authors found that tumors of vancomycin-treated mice were enriched with endogenous CD8+ T cells and CD11b+ CD103+ dendritic cells. Additionally, markers of T cell activation and cytotoxicity, such as granzyme B, perforin 1, and interferon γ (IFNγ), were increased, along with upregulation of factors involved in the antigen presentation pathway. These data suggested that endogenous CD8+ T cells and dendritic cells might potentiate CART activity via the cross-presentation of tumor-associated antigens, and was further confirmed by the fact that the administration of an anti-MHC-I blocking antibody abrogated these from the of preclinical mice of their gut microbiota fecal transplantation from human with lymphoma cells and treated with CART and oral tumor higher of dendritic cells, and markers were found in mice exposed to CART and vancomycin compared with the Moreover, Ruminococcaceae and Lachnospiraceae were decreased along with and which have been shown to impair antigen presentation and CD8+ T cell studies have reported different effects, activity on and In a retrospective Uribe-Herranz et al also that B-ALL patients receiving oral vancomycin for higher CART cell patients not exposed to this Despite the small these preliminary data demonstrate that the gut microbiota the immune by CART and the endogenous immune antitumor therapeutic strategies have been developed to modulate the microbiota activity and the antitumor effects of such as fecal microbiota transplantation and in therapy 1). The results achieved in patients treated with and allo-HCT suggest a role of these in CART as results of clinical in CART-treated patients are still of the of a complex and of from a for the absence of to a The its is that the of gut to the restoration of of the including and with the immune has been for the treatment of and has recently shown results both in and with acute GVHD after allogeneic clinical studies are its role as an of ICB, in GVHD and both in and and Despite the have recently due to the potential of and such as the of control and at large strategies to enhance CART immunotherapy by the gut CART = chimeric antigen receptor T cell therapy; = PIM = piperacillin-tazobactam, imipenem, meropenem; = is the use of that administration of specific bacterial or of to modulate the Despite the results achieved in disease and metabolic the effects of in with immunotherapy for are still the et al have reported antitumor effects of Bifidobacterium and in mice treated with ICB, due to the of T cells in the tumor the et al did not better survival and response in melanoma patients treated with and exposed to Moreover, they that mice and Bifidobacterium longum and tumor the mice and a significantly reduced of CD8+ T cells in tumor To the of and the potential detrimental effect of the first trial of a as a for has been published recently, its and the between and clinical response could not be assessed in this These the for further and clinical are to the potential of immunotherapy with and and possible to modulate the gut microbiota is the administration of that the and metabolism of bacteria in the such as and The clinical administration of an and to allo-HCT recipients, from the to day has to and acute GVHD by bacterial on the and the the a and immune or enhance the of CD8+ T In a GVHD preclinical directly the T cells the mice with T cells of markers of abundance of the and Blautia, and experienced GVHD the as the result of of the T the survival and of T cells, and antigen presentation in and with the activity in the effects of vancomycin with and CART data that a gut the tumor-associated and T cell in the the of reduced activity and promoted of T cells, in tumor to their to modulate T cell and the of via the such as and have also been investigated as during the T cell a more expression in et al demonstrated that patients with progression-free survival and higher response over with which correlated with alpha diversity and Ruminococcaceae and Faecalibacterium Furthermore, et al reported that patients on higher and achieved antitumor immune and reduced after the and its have been shown to the of and Bifidobacterium in a to previous a high abundance of Bifidobacterium enhanced the antitumor activity of the the activation of the endogenous dendritic cells and CD8+ Overall, and may impact and enhance the antitumor T cell response in or could be in the in Since no clinical data are the potential role of approaches in CART-treated patients be investigated in clinical the microbiota composition and are commonly used in hematological patients, both as and antibiotics cause of obligate anaerobes and gut the efficacy of immunotherapy or associated with toxicities. they have been associated with a high rate after allogeneic reduced antitumor efficacy of more recently, reduced survival in CART-treated treatment of the broad-spectrum could minimize the dysbiosis and the immune response against but the of antibiotics is Interestingly, to the effects of different antibiotics on the microbiota composition, a II clinical trial is in allo-HCT the effects of piperacillin-tazobactam cefepime, as a In preclinical and clinical studies have demonstrated that the gut microbiota a role in the response to the mechanisms is still a Furthermore, due to the high rate of the efficacy of CART is a on Stein-Thoeringer et pectinoschiza and Akkermansia muciniphila might be to whereas could be to T cells and their both during the in and the in In addition, a or would be significant in metabolism and gut microbiota a and a role in the gut microbiota and their impact on post-CART survival a treatment to the alpha diversity and its effect on studies are needed to confirm the effects of oral vancomycin in CART In a could enhance the presentation by endogenous cells and CART survival in the results in allo-HCT and patients, may be a of interest in CART-treated patients, in most exposed to Noteworthy, the impact of antibiotics and dysbiosis on CART to be In the gut microbiota has demonstrated a influence on therapy and immune data have suggested its potential role in the effects of CAR T cell but the mechanisms are not In this we have highlighted several potential therapeutic strategies to enhance the efficacy of engineered T cells and the of CART-treated patients by the potential of the gut are needed to the efficacy of these and achieve more to the and the and to of the and the the before to the of the of the and the before The authors have no of interest to The authors no of