ABSTRACT:Antibiotic (ABX)-induced microbiome dysbiosis is widespread in oncology, adversely affecting outcomes and side effects of various cancer treatments, including immune checkpoint inhibitors and chimeric antigen receptor T-cell (CAR-T) therapies. In this study, we observed that prior exposure to broad-spectrum ABXs with extended anaerobic coverage such as piperacillin-tazobactam and meropenem was associated with worse anti-CD19 CAR-T therapy survival outcomes in patients with large B-cell lymphoma (N = 422) than other ABX classes. In a discovery subset of these patients (n = 67), we found that the use of these ABXs was in turn associated with substantial dysbiosis of gut microbiome function, resulting in significant alterations of the gut and blood metabolome, including microbial effectors such as short-chain fatty acids (SCFAs) and other anionic metabolites, findings that were largely reproduced in an external validation cohort (n = 58). Broader evaluation of circulating microbial metabolites revealed reductions in indole and cresol derivatives, as well as trimethylamine N-oxide, in patients who received ABX treatment (discovery, n = 40; validation, n = 28). These findings were recapitulated in an immune-competent CAR-T mouse model, in which meropenem-induced dysbiosis led to a systemic dysmetabolome and decreased murine anti-CD19 CAR-T efficacy. Furthermore, we demonstrate that SCFAs can enhance the metabolic fitness of CAR-Ts, leading to improved tumor killing capacity. Together, these results suggest that broad-spectrum ABX deplete metabolically active commensals whose metabolites are essential for enhancing CAR-T efficacy, shedding light on the intricate relationship between ABX exposure, microbiome function and their impact on CAR-T efficacy. This highlights the potential for modulating the microbiome to augment CAR-T immunotherapy. This trial was registered at www.clinicaltrials.gov as #NCT06218602.

Immune deficits after CD19 chimeric antigen receptor (CAR) T-cell therapy can be long-lasting, predisposing patients to infections and non-relapse mortality. In B-cell non-Hodgkin lymphoma (B-NHL), the prognostic impact of immune reconstitution (IR) remains ill-defined, and detailed cross-product comparisons have not been performed to date. In this retrospective observational study, we longitudinally characterized lymphocyte subsets and immunoglobulin levels in 105 B-NHL patients to assess patterns of immune recovery arising after CD19 CAR-T. Three key IR criteria were defined as CD4+ T helper (TH) cells > 200/µL, any detectable B cells, and serum immunoglobulin G (IgG) levels >4 g/L. After a median follow-up of 24.6 months, 38% of patients displayed TH cells, 11% showed any B cells, and 41% had IgG recovery. Notable product-specific differences emerged, including deeper TH cell aplasia with CD28z- versus longer B-cell aplasia with 41BBz-based products. Patients with any IR recovery experienced extended progression-free survival (PFS) (median 20.8 vs. 1.7 months, p < 0.0001) and overall survival (OS) (34.9 vs. 4.0 months, p < 0.0001). While landmark analysis at 90 days confirmed improved PFS in patients with any recovery (34.9 vs. 8.6 months, p = 0.005), no significant OS difference was noted. Notably, 72% of patients with refractory disease never displayed recovery of any IR criteria. Early progressors showed diminished IR at the time of progression/relapse compared to patients with late progression/recurrence (after Day 90). Our results highlight the profound immune deficits observed after CD19 CAR-T and shed light on the intersection of IR and efficacy in B-NHL. Importantly, IR was impaired considerably postprogression, carrying significant implications for subsequent T-cell-engaging therapies and treatment sequencing.

BACKGROUND:Despite revolutionary efficacy of CD19-CAR-T cell therapy (CAR-T) in aggressive B cell lymphoma, many patients still relapse mostly early. In early failure, distinct drugs support CAR-T which makes reliable and early prediction of imminent relapse/refractoriness critical. A complete metabolic remission (CR) on Fluor-18-Deoxyglucose (FDG) Positron-Emission-Computed Tomography (PET) 30 days after CAR-T (PET30) strongly predicts progression-free survival (PFS), but still fails in a relevant proportion of patients. We aimed to identify additional routine parameters in PET evaluation to enhance CAR-T response prediction. RESULTS:Thirty patients with aggressive B cell lymphoma treated with CAR-T were retrospectively analyzed. Pre-CAR-T, LDH was the strongest PFS-predictor also by multivariate analysis. Post-CAR-T, 10 out of 14 patients (71.4%) with PET30-CR remained in disease remission, while 12 out of 16 patients (75%) with incomplete metabolic remission (PET30-nCR) relapsed after CAR-T. 28.6% of patients with PET30-CR ultimately progressed. Change of liver FDG-uptake from baseline to day30 (Delta-Liver-SUVmean) was identified as an independent biomarker for response. PET30-nCR and a decrease of Delta-Liver-SUVmean were associated with a high risk of tumor progression (HR 4.79 and 3.99, respectively). The combination of PET30 and Delta-Liver-SUVmean identified patients at very low, at intermediate and at very high risk of relapse (PFS not reached, 7.5 months, 1.5 months, respectively). CONCLUSION:Additionally to PET30 metabolic remission, longitudinal metabolic changes in Delta-Liver-SUVmean predicted CAR-T efficiency. Our results may guide early intervention studies aiming to enhance CAR-T particularly in the very high-risk patients.

Introduction: Emerging evidence suggests that immune dysregulation drives resistance to cellular therapies. In particular, low CAR T-cell expansion and inferior patient outcomes have been associated with an inflamed immune milieu. Here, we investigated whether myeloid cells and serum proteomics are linked to T-cell dysfunction and treatment failure. Methods: Patients with r/r B-NHL who underwent treatment with axi-cel, brexu-cel, or tisa-cel in the third- or later-line setting were retrospectively included (n=74). Aliquots of EDTA-anticoagulated peripheral blood and serum were collected. Immune checkpoint (IC) expression (PD-1, TIM-3, and LAG-3) was assessed through flow cytometry before CAR T-cell infusion (time of leukapheresis, day -5 and day 0) as a surrogate for T-cell exhaustion. More specifically, the abundance of different co-expression profiles of ICs was compared and defined as non-exhausted (PD-1-TIM-3-LAG-3-), progenitor-exhausted (PD-1+TIM-3+LAG-3-), and terminally-exhausted (PD-1+TIM-3+LAG-3+). Using the Olink® Immuno-Oncology panel, 92 immune-related proteins were measured at day 0. A next-generation single-cell proteogenomics approach (BD RhapsodyTM) was applied to analyze the transcriptome of myeloid cells before and after infusion (day -5 and day 7). Responding patients (R, complete or partial remission) were compared to non-responding patients (NR, stable or progressive disease) according to 3-month (PET-) CT scans. Results: At baseline, NR showed lower frequencies of non-exhausted (apheresis, p = 0.025; day -5, p = 0.0236; day 0, p = 0.0496) and higher frequencies of terminally-exhausted T-cells (-5, p = 0.0053) compared to R. Interestingly, these surrogates for pre-existing T-cell dysfunction in NR were linked to an inflammatory state. Patients with low levels of non-exhausted T-cells also had higher levels of CRP (p = 0.0059), Ferritin (p = 0.0333), and LDH (p = 0.0268) compared to patients with high levels of non-exhausted T-cells before infusion. Network analysis correlating the abundance of immune-modulatory proteins with baseline IC expression on T cells revealed numerous connections for NR but merely associations for R, suggesting an impact of the serum protein milieu on IC expression in NR. We dissected these networks in terms of the degree centrality differences for the different proteins (i.e., the variations in the number of connections among the nodes between NR and R). While we identified a higher degree centrality for co-stimulatory proteins such as IL-18, CD8A and ICOSLG in R, we noted higher degree centrality for immune-inhibitory and tumor-related proteins such as CXC3L1, TNFRSF21, VEGFA and IL-6 in NR. Accordingly, we linked a higher abundance of CD8A and a lower abundance of TNFRSF21 and IL-6 to the frequency of non-exhausted T-cells at baseline using a linear regression model. Finally, we asked whether the myeloid compartment at baseline drives these differences in the serum protein milieu observed between NR and R. We identified four phenotypic and transcriptomic different monocytic and dendritic cell populations. Interestingly, two classical monocyte-like populations (CD14+CD16+HLA-DRlo) showed divergent population dynamics in NR but not R when assessing pre- and post-infusion time points. Notably, gene set enrichment analysis revealed an upregulation of immunosuppressive pathways, such as genes regulated by NF-KB in response to TNF, in several myeloid populations in NR but not R (classical monocyte-like 2, p < 0.0001; non-classical monocyte-like, p < 0.0001; DC-like, p = 0.0024). Surprisingly, monocytic populations in R revealed an upregulation of genes with immune stimulatory functions linked to responses to interferon-alpha or -gamma proteins (classical monocyte-like 1 and 2, p = 0.0058 and p = 0.0003; non-classical monocyte-like, p = 0.0003). Conclusion: These data suggest that CAR-T non-responders exhibit pre-existing T-cell dysfunction resulting from a systemic inflamed proteomic and cellular environment. Notably, the profound alterations within the myeloid compartment highlight diverging immune-modulating functions in non-responding and responding B-NHL patients. These findings set the further scope for scientific investigations not only to mitigate immune dysregulation but also to target the innate immune compartment for enhanced CAR T-cell responses.

Introduction The gut microbiome plays an important role in modulating the host's immune system. Multiple clinical studies have linked gut microbiome dysbiosis to poorer clinical outcomes in patients undergoing adoptive immune-cell therapies (Stein-Thoeringer et al. Science 2019, Stein-Thoeringer et al. Nature Medicine 2023). Gut microbiome dysbiosis has been linked in particular to the intestinal expansion of Enterococcus spp., often due to antibiotic pretreatments. This expansion was associated with altered immune responses and pathologies such as GVHD (Stein-Thoeringer et al. Science 2019; Taur et al. Clin Infect Dis 2012). Here, we hypothesized that Enterococcus spp. may also impact the outcomes of CD19-CAR-T cell therapy as these patients are highly susceptible to antibiotic-induced gut dysbiosis. Methods 59 patients with B cell malignancies who underwent treatment with CD19-CAR-T cells at two university cancer centers in Germany (Munich and Heidelberg) were included, where we collected serial clinical data and stool samples. Enterococcus spp. were isolated from stool specimens and identified via Sanger sequencing. In our downstream analyses, we only focused on samples with E. faecalis or E. faecium and carried out several in vitro functional tests. Antibiotic resistance was determined using disk diffusion assays. Immunogenicity and strain virulence was determined by measuring cytokine responses of enterococcal lysates pulsed on human PBMCs from healthy donors and by quantitative testing for biofilm formation. Metabolic characteristics were assessed by recording growth rates in a minimal medium while adding different carbohydrates. All stool samples were sequenced by shotgun sequencing. In addition, Enterococcus spp. isolated from 42 LMU patients were individually sequenced by whole genome sequencing to determine antimicrobial resistance genes and virulence factors. Results In vitro testing showed an increase in ampicillin resistance for E. faecium and E. faecalis from pre- to post-CAR-T time points (Fisher's exact test p-value = 0.08754 and p-value = 0.06234, respectively). This increase was particularly evident in the Heidelberg patient cohort for E. faecalis (Fisher's exact test p-value = 0.02105) and in the Munich patient cohort for E. faecium (Fisher's exact test p-value = 0.07337). In addition, we observed an increased imipenem resistance for E. faecalis (Fisher's exact test p-value = 0.0534) and, specifically for E. faecium (Fisher's exact test p-value = 0.05235), in the Heidelberg cohort. These results suggest a general increase in resistance to beta-lactam antibiotics after cell therapy, most likely due to intense antibiotic treatments. When assessing strain virulence in a biofilm formation assay, we detected higher biofilm formation in E. faecium isolated from responder patients (response measured at day 180; Wilcoxon test p-value = 0.03). Notably, interleukin-6 (IL-6) release from stimulated PBMCs was significantly correlated with biofilm formation (p-value ≤ 0.001) and significantly higher in Enterococcus spp. isolates from patients with high-grade ICANS (Wilcoxon test p-value = 0.0054). The association of treatment response and microbial virulence, measured through IL-6 release and biofilm formation, could imply an increased immune activation in patients, possibly resulting in a greater CAR-T cell activity. Strains of E. faecalis that induced high IL-6 release also showed a higher capacity for metabolizing carbohydrates, i.e. glucose, fructose and lactose (Wilcoxon test p-value = 0.0016, p-value = 0.00052 and p-value = 0.019, respectively), which further supports elevated virulence. Ongoing whole genome sequencing of the isolates aims to characterize the genomic correlates of virulence driving the immunogenic phenotype of the identified strains and will be presented. Conclusions Our novel findings indicate that antibiotic resistance, metabolic phenotypes and, notably, virulence of Enterococcus spp. can significantly affect the host on a strain-specific level. These findings suggest the need to perform strain-specific phenotyping in future microbe - host interaction studies in patients undergoing cell-based immunotherapies. Ultimately enabling future microbiome-based patient assessment and monitoring.

Bridging therapy (BT) after leukapheresis is required in most relapsed/refractory (R/R) large B-cell lymphoma (LBCL) patients receiving chimeric antigen receptor (CAR) T cells. Bendamustine-containing regimens are a potential BT option. We aimed to assess if this agent had a negative impact on CAR-T outcomes when it was administered as BT. We included R/R LBCL patients from six centers who received systemic BT after leukapheresis from February 2019 to September 2022; patients who only received steroids or had pre-apheresis bendamustine exposure were excluded. Patients were divided into two BT groups, with and without bendamustine. Separate safety and efficacy analyses were carried out for axi-cel and tisa-cel. Of 243 patients who received BT, bendamustine (benda) was included in 62 (26%). There was a higher rate of BT progressors in the non-benda group (62% vs. 45%, p = 0.02). Concerning CAR-T efficacy, complete responses were comparable for benda versus non-benda BT cohorts with axi-cel (70% vs. 53%, p = 0.12) and tisa-cel (44% vs. 36%, p = 0.70). Also, 12-month progression-free and overall survival were not significantly different between BT groups with axi-cel (56% vs. 43% and 71% vs. 63%) and tisa-cel (25% vs. 26% and 52% vs. 48%); there were no differences when BT response was considered. CAR T-cell expansion for each construct was similar between BT groups. Regarding safety, CRS G ≥3 (6% vs. 6%, p = 0.79), ICANS G ≥3 (15% vs. 17%, p = 0.68), severe infections, and neutropenia post-infusion were comparable among BT regimens. BT with bendamustine-containing regimens is safe for patients requiring disease control during CAR T-cell manufacturing.

Infections account for the highest number of non-relapse-related deaths after CAR T cell therapy, underscoring the necessity for clinical risk-mitigating strategies and a deeper understanding of CAR T cell-related cytopenia.

CD19 specific chimeric antigen receptor (CAR) T-cell therapy with patient-derived T cells has emerged as novel and effective treatment for relapsed or refractory (r/r) lymphoma. Imaging biomarkers are becoming increasingly important in risk assessment. Recently, the International Prognostic Metabolic Index (IMPI) consisting of metabolic tumor volume (MTV), patient age and Ann Arbor stage was introduced for prognostic assessment in NHL. Since higher patient age is a favorable prognostic factor and a high MTV is a negative prognostic factor in the context of CART, we compared the predictive value of the individual components of the IMPI for progression-free survival (PFS) and overall survival (OS). Consecutive r/r NHL patients treated with CD19 CAR T-cell therapy and 18F FDG-PET/CT imaging at baseline less than 2 weeks before lymphodepletion and infusion of CAR T cells were included. Patient age was calculated at the time of lymphodepletion. Ann Arbor stages were determined at the baseline PET/CT. MTV was also quantified on the same PET/CT scan before CART and quantified with LIFEx software using semi-automated segmentation with an absolute SUV cutoff of 4 defining metabolic active lymphoma. Survival analysis of PFS and OS was analyzed and plotted with Kaplan-Meier curves. Out of all examined patients 43 patients were included (37% female, 63% male) with a median age of 66 years and a median baseline MTV of 276 mL. The Ann Arbor stage was I in 4 patients, II in 11 patients, III in 8 patients and IV in 4 patients. The distribution of patients in to two different groups by median IMPI showed minor, non-significant differences in median PFS (97 vs 364 days; p=0.198) and OS (657 days vs not reached; p=0.718). Dichotomization by median MTV alone showed a larger, statistically significant difference (p=0.037) in median PFS (87 vs 659 days) and a larger, non-significant difference in OS (p=0.300). Patients with older age had slightly longer PFS (153 vs 97 days) and a comparable OS (686 vs 657 days), but these findings were not statistically significant (both p>0.05). In addition, there were no relevant differences when the patients were divided into subgroups according to their Ann Arbor stages. For r/r NHL patients on CART, the IMPI is a promising tool. However, in our study, the MTV alone was superior in estimating prognosis. Future studies with a larger cohort should investigate the best combination of the individual components. The integration of multiple diagnostic modalities as in IMPI offers a promising method to clinically assess patient risk in CART treated lymphoma patients and should be further investigated.

Sex differences in the incidence and mortality are evident in a wide range of cancers. This is not only due to sex-related variations in behavioral cancer risk factors, but also due to biological and molecular effects of sexual differentiation. Focusing on immunotherapeutic approaches like immune checkpoint inhibition or allogeneic stem cell transplantation, several reports have associated male sex with inferior outcomes. However, the exact impact remains controversial, and underlying mechanisms are incompletely understood. Here, we sought to explore the effects of the patient's biological sex on the outcomes of CD19 CAR-T cell therapy. To this end, we retrospectively analyzed disease and laboratory features together with survival in a cohort of 214 patients treated with axicabtagene ciloleucel (Axi-cel) for relapsed/refractory large B-cell lymphoma (LBCL) in the third- or later-line setting at four CAR-T centers. Predictors of progression-free survival (PFS) were analyzed by uni- and multivariate comparisons. The analyzed patient cohort comprised 119 male and 95 female patients, with a median age of 64 years (range 19-79). Most baseline patient characteristics were not significantly different for male and female patients (see attached table). Ferritin levels prior to lymphodepletion, however, were significantly higher in males (median 505 ng/ml vs. 325 ng/ml in females, p=0.025). The incidences of ASTCT grade ≥ 3 cytokine release syndrome (CRS) and immune effector cell associated neurotoxicity syndrome (ICANS) were not significantly different between females and males. However, objective response rates were significantly higher in female patients (88% vs. 75% in males, p=0.03). Additionally, females had a significantly superior PFS compared to males (median PFS not reached [95% CI 12.2 - NA months] in females vs. 4.2 months [95% CI 3.2 - 9.2 months] in males, Hazard ratio 1.8 [1.2 - 2.6], p=0.005, 1-year PFS estimate 61% in females vs. 39% in males). Overall survival (OS) was also significantly longer in females (median OS not reached in females vs. 22.6 months [95% CI 13.3 months - not reached] in males, Hazard ratio 1.7 [1.1 - 2.9], p=0.028). We confirmed the relevance of the patient's sex in multivariate analysis. We used Lasso modelling of variables with significant (p<0.05) association with PFS from univariate Cox regression analyses (ECOG, LDH, CRP and ferritin levels, Ann Arbor stage, bulky or extranodal disease [END], No. of therapy lines prior to CAR-T cell therapy and response to bridging therapy). Male sex, non-response to bridging, stage III/IV disease, END and No. of therapy lines remained independent risk factors for inferior PFS (Lasso coefficients >0.1), with male sex and non-response to bridging having the most significant effect (Lasso coefficient 0.54 for both variables). Finally, employing logistic regression of the independent variables from multivariate analysis, propensity score matching was used to better delineate the effect of sex on PFS. Sufficient balance was achieved between male and female patients, with all standardized mean differences below 0.12 after matching. In the matched analysis, PFS remained significantly superior for female patients (1-year PFS estimate 61% in females vs. 40% in males, log-rank p=0.008). For OS, the observed difference was of borderline significance in the matched dataset (1-year OS estimate 77% in females vs. 61% in males, log-rank p=0.051). Taken together, we demonstrate that female patients have significantly superior outcomes following Axi-cel therapy for r/r LBCL, even after careful adjustment for variables known to impact treatment results. A deeper understanding of the underlying mechanisms of these outcome disparities might enable the optimization of T cell-based treatment platforms in the future, both in female and male patients. Male patients may potentially benefit from intensified CAR-T (combination) treatment strategies.

Prolonged cytopenias after chimeric antigen receptor (CAR) T cell therapy are a significant clinical problem and the underlying pathophysiology remains poorly understood. Here, we investigated how (CAR) T cell expansion dynamics and serum proteomics affect neutrophil recovery phenotypes after CD19-directed CAR T cell therapy. Survival favored patients with "intermittent" neutrophil recovery (e.g., recurrent neutrophil dips) compared to either "quick" or "aplastic" recovery. While intermittent patients displayed increased CAR T cell expansion, aplastic patients exhibited an unfavorable relationship between expansion and tumor burden. Proteomics of patient serum collected at baseline and in the first month after CAR-T therapy revealed higher markers of endothelial dysfunction, inflammatory cytokines, macrophage activation, and T cell suppression in the aplastic phenotype group. Prolonged neutrophil aplasia thus occurs in patients with systemic immune dysregulation at baseline with subsequently impaired CAR-T expansion and myeloid-related inflammatory changes. The association between neutrophil recovery and survival outcomes highlights critical interactions between host hematopoiesis and the immune state stimulated by CAR-T infusion.

Chimeric antigen receptor T-cell therapy (CART) can be administered outpatient yet requires management of potential side effects such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). The pre-infusion tumor burden is associated with CRS, yet there is no data on the relevance of pre-infusion tumor growth rate (TGR). Our objective was to investigate TGR for the occurrence and severity of CRS and ICANS. Consecutive patients with available pre-baseline and baseline (BL) imaging before CART were included. TGR was determined as both absolute (abs) and percentage change (

Background aims: Chimeric antigen receptor T-cell therapy (CART) prolongs survival for patients with refractory or relapsed lymphoma, yet its efficacy is affected by the tumor burden. The relevance of tumor kinetics before infusion is unknown. We aimed to study the prognostic value of the pre-infusion tumor growth rate (TGRpre-BL) for progression-free (PFS) and overall survival (OS).Methods: Consecutive patients with available pre-baseline (pre-BL) and baseline (BL) computed tomography or positron emission tomography/computed tomography scan before CART were included. TGR was determined as change of Lugano criteria-based tumor burden between pre-BL, BL and follow-up examinations (FU) in relation to days between imaging exams. Overall response rate (ORR), depth or response (DoR) and PFS were determined based on Lugano criteria. Multivariate regression analysis studied association of TGR with ORR and DoR. Proportional Cox regression analysis studied association of TGR with PFS and OS.Results: In total, 62 patients met the inclusion criteria. The median TGRpre-BL was 7.5 mm2/d (interquartile range -14.6 mm2/d to 48.7 mm2/d); TGRpre-BL was positive (TGRpre-BL POS) in 58% of patients and negative (TGRpre-BL NEG, indicating tumor shrinkage) in 42% of patients. Patients who were TGRpre-BL POS had a 90-day (FU2) ORR of 62%, a DoR of -86% and a median PFS of 124 days. Patients who were TGRpre-BL NEG had a 90day ORR of 44%, DoR of -47% and a median PFS of 105 days. ORR and DoR were not associated with slower TGR (P = 0.751, P = 0.198). Patients with an increase of TGR from pre-BL over BL to 30-day FU (FU1) & GE;100% (TGRpre-BL-to-FU1 & GE;100%) showed a significant association with shorter median PFS (31 days versus 343 days, P = 0.002) and shorter median OS after CART (93 days versus not reached, P < 0.001), compared with patients with TGRpre-BL-to-FU1<100%.Conclusions: In the context of CART, differences in pre-infusion tumor kinetics showed minor differences in ORR, DoR, PFS and OS, whereas the change of the TGR from pre-BL to 30-day FU significantly stratified PFS and OS. In this patient population of refractory or relapsed lymphomas, TGR is readily available based on pre-BL imaging, and its change throughout CART should be explored as a potential novel imaging biomarker of early response.& COPY; 2023 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.

BackgroundChimeric antigen receptor T-cell therapy (CART) prolongs survival for patients with refractory or relapsed lymphoma. Discrepancies among different response criteria for lymphoma under CART were recently shown. Our objective was to evaluate reasons for discordance among different response criteria and their relation to overall survival.MethodsConsecutive patients with baseline and follow-up imaging at 30 (FU1) and 90 days (FU2) after CART were included. Overall response was determined based on Lugano, Cheson, response evaluation criteria in lymphoma (RECIL) and lymphoma response to immunomodulatory therapy criteria (LYRIC). Overall response rate (ORR) and rates of progressive disease (PD) were determined. For each criterion reasons for PD were analyzed in detail.Results41 patients were included. ORR was 68%, 68%, 63%, and 68% at FU2 by Lugano, Cheson, RECIL, and LYRIC, respectively. PD rates differed among criteria with 32% by Lugano, 27% by Cheson, 17% by RECIL, and 17% by LYRIC. Dominant reasons for PD according to Lugano were target lesion (TL) progression (84.6%), new appearing lesions (NL; 53.8%), non-TL progression (27.3%), and progressive metabolic disease (PMD; 15.4%). Deviations among the criteria for defining PD were largely explained by PMD of preexisting lesions that are defined as PD only by Lugano and non-TL progression, which is not defined as PD by RECIL and in some cases classified as indeterminate response by LYRIC.ConclusionsFollowing CART, lymphoma response criteria show differences in imaging endpoints, especially in defining PD. The response criteria must be considered when interpreting imaging endpoints and outcomes from clinical trials.

Early fever after chimeric antigen receptor T-cell (CAR-T) therapy can reflect both an infection or cytokine release syndrome (CRS). Identifying early infections in the setting of CRS and neutropenia represents an unresolved clinical challenge. In this retrospective observational analysis, early fever events (day 0–30) were characterized as infection versus CRS in 62 patients treated with standard-of-care CD19.CAR-T for relapsed/refractory B-cell non-Hodgkin lymphoma. Routine serum inflammatory markers (C-reactive protein [CRP], interleukin-6 [IL-6], procalcitonin [PCT]) were recorded daily. Exploratory plasma proteomics were performed longitudinally in 52 patients using a multiplex proximity extension assay (Olink proteomics). Compared with the CRSonly cohort, we noted increased event-day IL-6 (median 2243 versus 64 pg/mL, P = 0.03) and particularly high PCT levels (median 1.6 versus 0.3 µg/L, P < 0.0001) in the patients that developed severe infections. For PCT, an optimal discriminatory threshold of 1.5 µg/L was established (area under the receiver operating characteristic curve [AUCROC] = 0.78). Next, we incorporated day-of-fever PCT levels with the patient-individual CAR-HEMATOTOX score. In a multicenter validation cohort (n = 125), we confirmed the discriminatory capacity of this so-called HT10 score for early infections at first fever (AUCROC = 0.87, P < 0.0001, sens. 86%, spec. 86%). Additionally, Olink proteomics revealed pronounced immune dysregulation and endothelial dysfunction in patients with severe infections as evidenced by an increased ANGPT2/1 ratio and an altered CD40/CD40L-axis. In conclusion, the high discriminatory capacity of the HT10 score for infections highlights the advantage of dynamic risk assessment and supports the incorporation of PCT into routine inflammatory panels. Candidate markers from Olink proteomics may further refine risk-stratification. If validated prospectively, the score will enable risk-adapted decisions on antibiotic use.

Increasing evidence suggests that the gut microbiome may modulate the efficacy of cancer immunotherapy. In a B cell lymphoma patient cohort from five centers in Germany and the United States (Germany, n = 66; United States, n = 106; total, n = 172), we demonstrate that wide-spectrum antibiotics treatment (‘high-risk antibiotics’) prior to CD19-targeted chimeric antigen receptor (CAR)-T cell therapy is associated with adverse outcomes, but this effect is likely to be confounded by an increased pretreatment tumor burden and systemic inflammation in patients pretreated with high-risk antibiotics. To resolve this confounding effect and gain insights into antibiotics-masked microbiome signals impacting CAR-T efficacy, we focused on the high-risk antibiotics non-exposed patient population. Indeed, in these patients, significant correlations were noted between pre-CAR-T infusion Bifidobacterium longum and microbiome-encoded peptidoglycan biosynthesis, and CAR-T treatment-associated 6-month survival or lymphoma progression. Furthermore, predictive pre-CAR-T treatment microbiome-based machine learning algorithms trained on the high-risk antibiotics non-exposed German cohort and validated by the respective US cohort robustly segregated long-term responders from non-responders. Bacteroides , Ruminococcus , Eubacterium and Akkermansia were most important in determining CAR-T responsiveness, with Akkermansia also being associated with pre-infusion peripheral T cell levels in these patients. Collectively, we identify conserved microbiome features across clinical and geographical variations, which may enable cross-cohort microbiome-based predictions of outcomes in CAR-T cell immunotherapy.

Background Chimeric antigen receptor T-cell therapy (CART) is effective for patients with refractory or relapsed lymphoma with prolongation of survival. We aimed to improve the prediction of Lugano criteria for overall survival (OS) at 30-day follow-up (FU1) by including the pre-infusion tumor growth rate (TGRpre-BL) and its early change to 30-day FU1 imaging (TGRpost-BL).Methods Consecutive patients with pre-baseline (pre-BL), baseline (BL) and FU1 imaging with CT or positron emission tomography/CT before CART were included. TGR was defined as change of Lugano criteria-based tumor burden between pre-BL, BL and FU1 examinations in relation to days between imaging examinations. Overall response and progression-free survival were determined based on Lugano criteria. Proportional Cox regression analysis studied association of TGR with OS. For survival analysis, OS was analyzed using Kaplan-Meier survival curves.Results Fifty-nine out of 81 patients met the inclusion criteria. At 30-day FU1 8 patients (13.6%) had a complete response (CR), 25 patients (42.4%) a partial response (PR), 15 patients (25.4%) a stable disease (SD), and 11 patients (18.6%) a progressive disease (PD) according to CT-based Lugano criteria. The median TGRpre-BL was −0.6 mm2/day, 24.4 mm2/day, −5.1 mm2/day, and 18.6 mm2/day and the median TGRpost-BL was −16.7 mm2/day, −102.0 mm2/day, −19.8 mm2/day and 8.5 mm2/day in CR, PR, SD, and PD patients, respectively. PD patients could be subclassified into a cohort with an increase in TGR (7 of 11 patients (64%), PD TGRpre-to-post-BL INCR) and a cohort with a decrease in TGR (4 of 11 patients (36%), PD TGRpre-to-post-BL DECR) from pre-BL to post-BL. PD TGRpre-to-post-BL DECR patients exhibited similar OS to patients classified as SD, while PD TGRpre-to-post-BL INCR patients had significantly shorter OS (65 days vs 471 days, p<0.001).Conclusion In the context of CART, the additional use of TGRpre-BL and its change to TGRpost-BL determined at 30-day FU1 showed better OS prognostication for patients with overall PD according to Lugano criteria. Therefore, this modification of the Lugano classification should be explored as a potential novel imaging biomarker of early response and should be validated prospectively in future studies.

PURPOSE Approximately 30%-40% of patients with relapsed/refractory (R/R) large B-cell lymphoma (LBCL) infused with CD19-targeted chimeric antigen receptor (CAR) T cells achieve durable responses. Consensus guidelines suggest avoiding bendamustine before apheresis, but specific data in this setting are lacking. We report distinct outcomes after CAR T-cell therapy according to previous bendamustine exposure. METHODS The study included CAR T-cell recipients from seven European sites. Safety, efficacy, and CAR T-cell expansion kinetics were analyzed according to preapheresis bendamustine exposure. Additional studies on the impact of the washout period and bendamustine dose were performed. Inverse probability treatment weighting (IPTW) and propensity score matching (PSM) analyses were carried out for all efficacy comparisons between bendamustine-exposed and bendamustine-naïve patients. RESULTS The study included 439 patients with R/R LBCL infused with CD19-targeted commercial CAR T cells, of whom 80 had received bendamustine before apheresis. Exposed patients had significantly lower CD3 + cells and platelets at apheresis. These patients had a lower overall response rate (ORR, 53% v 72%; P < .01), a shorter progression-free survival (PFS, 3.1 v 6.2 months; P = .04), and overall survival (OS, 10.3 v 23.5 months; P = .01) in comparison with the bendamustine-naïve group. Following adjustment methods for baseline variables, these differences were mitigated. Focusing on the impact of bendamustine washout before apheresis, those with recent (<9 months) exposure (N = 42) displayed a lower ORR (40% v 72%; P < .01), shorter PFS (1.3 v 6.2 months; P < .01), and OS (4.6 v 23.5 months; P < .01) in comparison with bendamustine-naïve patients. These differences remained significant after IPTW and PSM analysis. Conversely, the cumulative dose of bendamustine before apheresis did not affect CAR-T efficacy outcomes. CONCLUSION Recent bendamustine exposure before apheresis was associated with negative treatment outcomes after CD19-targeted CAR T-cell therapy and should be therefore avoided in CAR T-cell candidates.

Introduction: Despite the curative potential of CAR T-cells, a significant number of patients fail to respond or relapse early. Recently, we and others showed that early expansion failure, and particularly CAR T-cell levels at day 7, are strong predictors for treatment resistance (Blumenberg et al, Blood Supplement 2022; Locke et al, Blood Adv 2020). Here we dissected early characteristics of CAR T-cell dysfunction based on immune checkpoint expression, transcriptomic signatures and cytokine profiles and investigated their link to CAR T-cell expansion failure and patient outcome of CD19-targeted CAR T-cell therapy. Methods: Patients with r/r B-NHL who underwent treatment with axi-cel or tisa-cel between January 2019 and November 2021 in the third- or later-line setting at the LMU in Munich were included (n=55). EDTA-anticoagulated peripheral blood and serum was collected. CD19 CAR and immune checkpoint expression (IC; PD-1, TIM-3, LAG-3 and CD224) was assessed by multiparameter flow cytometry on day 14 after CAR T-cell infusion. A next-generation targeted single-cell proteogenomics approach (BD Rhapsody TM) to analyze the transcriptome of distinct CAR T-cell subpopulations on day 7 and day 28 after CAR T-cell infusion. A total of 92 human immuno-oncology related proteins were simultaneously measured using the Olink® Immuno-Oncology panel on day 14 after infusion. Signatures of CAR T-cell dysfunction have been compared between patients with low (loEx) or high (hiEx; < vs ≥ 19 CAR T-cells/µl at day 7 post infusion)CAR T-cell expansion and were put into context of clinical outcome measures. Results: CAR T-cell levels at day 7 were inversely correlated to co-expression of PD-1 (r = -0.4923, p = 0.0011, n = 41), TIM-3 (r = -0.3634, p = 0.0195, n = 41) and LAG-3 (r = -0.5412, p = 0.0003, n = 41) on CAR T-cells at day 14, suggestive of an association of early expansion failure and dysfunction of CAR T-cells. Indeed, loEx (n=26) showed higher frequencies of CAR T-cells coexpressing several IC, including LAG-3 + (p=0.0036), LAG-3 +PD-1 + (p = 0.0048), or LAG-3 +TIM-3 + (p = 0.0065) compared to hiEx (n=15) on day 14 after transfusion. Conversely, we detected higher frequencies of non-dysfunctional CAR T-cell phenotypes negative for several IC, such as CD244 +PD-1 -TIM-3 - (p = 0.0006), CD244 +LAG-3 -PD-1 -TIM-3 - (p = 0.001) and CD244 +LAG-3 +PD-1 -TIM-3 - (p = 0.011) in hiEx. Next, we applied uniform manifold approximation and projection (UMAP) data to examine the surface expression of IC on CAR T-cells at day 14 post-infusion according to treatment response. Unsupervised clustering revealed a higher abundance of CAR T-cell clusters positive for several IC (CD244 +LAG-3 -PD-1 +TIM-3 +) in NR (n = 16) compared to R (n = 23). By contrast, CAR T-cell clusters containing CAR T-cells expressing no IC were more abundant in R compared to NR. In addition, we found significantly increased levels of a highly dysfunctional CAR T-cell phenotype positive for all four IC in NR compared to R at day 14 post infusion (p = 0.0178, n = 46). When specifically comparing the transcriptome of CAR T-cells between NR (n = 6) and R (n = 6), immune inhibitory and exhaustion-related genes were upregulated in NR, such as GZMK, KLRC1 and NR4A2 (figure 1, cluster 1). Conversely, CAR T-cells in R overexpressed genes related to T-cell effector function and cell differentiation, such as TIMP-1, TNFDF10 and MYC (figure 1, cluster 0). Consistent with these findings, a multiplexed proteomics assay for immunomodulatory serum factors in day 14 samples revealed an upregulation of proteins linked to an immune inhibitory and inflamed milieu in patients with highly dysfunctional CAR T-cells (e.g., sPD-L1 and sLAG-3, p = 0.0235 and p = 0.0264, respectively, n = 46) as well as in non-responding patients (e.g., sPD-L1 and IL-6, p = 0.0036 and p = 0.0053, respectively, n = 55). Conclusion: We were able to characterize a phenotypic, transcriptomic and proteomic signature of CAR T-cell dysfunction in post-infusion samples of both patients experiencing CAR T-cell expansion failure as well as lack of radiographic response. Our data show that treatment failure might be predicted not only by CAR T-cell kinetics but also by early assessment of the CAR T-cell phenotype, transcriptome and secretome, and thus enables identification of patients in need of salvage treatment.

Introduction: CD19 chimeric antigen receptor T-cell therapy (CD19.CAR-T) has profoundly improved treatment options for patients with relapsed/refractory B-cell lymphomas (r/r BCL), but is also associated with distinct toxicities. To elucidate host-intrinsic immunometabolic factors contributing to clinical benefit or immunotoxicity, we recently demonstrated that an increase of metabolically high-risk adipose tissue mass is associated with an elevated cytokine release syndrome (CRS) risk but also with improved survival (Haematologica 2022, CIR 2023). To gain further insights into the underlying immunometabolic mechanisms, we analyzed temporal dynamic changes of serum metabolites in r/r BCL patients during the first 14 days of CD19.CAR-T treatment. Methods: In this single-center analysis, we studied patients receiving CD19.CAR-T for r/r large BCL or mantle cell lymphoma (MCL) between 01/2019 and 12/2021 in a real-world setting. All patients were consecutively treated in our department (LMU University Hospital) and serum was prospectively collected in the morning hours from fasted patients beginning at the day of CAR-T infusion (D0), and 3-5 and 14 days later. Using liquid chromatography mass-spectrometry, we quantitatively analyzed a total of 305 water-soluble metabolites and 734 lipids. Metabolomic data was integrated with clinical patient records including CRS, 1-year progression-free survial (PFS) and amount of visceral adipose tissue (VAT). Results: In total, we measured 170 serum samples from 55 r/r LBCL and 4 r/r MCL patients, of which 52 had serial samples at all three time points (D0: 55, D3-5: 59, D14: 56). The following CAR-T products were administered: axi-cel (n = 26), tisa-cel (n = 29) and brexu-cel (n = 4). 25 (42.4%) patients developed a CRS grade ≥ 2, while 34 patients had a CRS grade 0-1. The best 90-day overall response rate was 52.5%, with a complete remission rate of 42.4%. At 1 year, 67.8% of patients progressed. From the 1039 metabolites measured, 525 metabolites passed quality control. Metabolite values were normalized and auto-scaled for further analysis. A partial least square differential analysis based on the collected time points led to a clear separation of samples with uric acid, desoxy-ribose-5-phosphate (dRibose-P), essential amino acids and (phospho)lipids having the strongest impact. Whereas 56 metabolites showed a significant increase from D0 to D14 led by uric and essential amino acids, 34 metabolites decreased over the course of treatment with the strongest reduction in dRibose-P and sphingosine phosphate. Performing a metabolic pathway enrichment analysis based on the most altered metabolite levels, we detected 22 significantly affected metabolic pathways and programs including aerobic glycolysis, urea cycle, amino acid and lipid metabolism (FDR < 0.1, Fig. 1A). To further delineate metabolite changes in regards to CRS, 1-year PFS and amount of VAT, we conducted a multivariate empirical bayes analysis of variance to specifically detect metabolites with differential temporal dynamics and considered all metabolites with an FDR < 0.1. Patients who developed a CRS ≥ 2° showed increased levels of kynurenine, phosphatidylethanolamides and lysophosphatidylserines, and decreased levels of ornithine and phosphatidylinositols. 1-year progressors showed a significant increase of several phosphatidylinositols beginning at D3-5 after CAR-T infusion, and decreased levels of phosphatidylcholines. For VAT, we used a threshold of 144 cm² which was derived from previously conducted computer tomography segmentation analyses. As expected, VAT cohorts mostly differed in fatty acid metabolism-associated metabolites including (acyl)carnitines, cholesterol esters, triglycerides and xanthine. In order to identify metabolites being associated with multiple outcomes, we looked for mutual metabolites among distinct cohort profiles. Here, one example is phosphatidylinositol-(22:5/16:0), which was increased in CRS 0-1° and 1y-progressors ( Fig. 1B). Conclusions: Our study highlights that CD19.CAR-T treatment is accompanied by broad changes in the serum metabolome of r/r BCL patients, which can be used to profile patients being affected by CAR-T-related immunotoxicity and their response to treatment. In ongoing analyses, we evaluate the use of these profiles for predictive models, and assess their impact on CAR-T cell function in-vitro.