Despite high response rates after initial chemotherapy in patients with acute myeloid leukemia (AML), relapses occur frequently, resulting in a five-year-survival by <30% of the patients. Hitherto, allogeneic hemotopoietic stem cell transplantation (allo-HSCT) is the best curative treatment option in intermediate and high risk AML. It is the proof-of-concept for T cell-based immunotherapies in AML based on the graft-versus-leukemia (GvL)-effect, but it also bears the risk of graft-versus-host disease. CD19-targeting therapies employing chimeric antigen receptor (CAR) T cells are a breakthrough in cancer therapy. A similar approach for myeloid malignancies is highly desirable. This article gives an overview on the state-of-the art of preclinical and clinical studies on suitable target antigens for CAR T cell therapy in AML patients.

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.

Background: T lymphocyte collection through leukapheresis is an essential step for chimeric antigen receptor T (CAR-T) cell therapy. Timing of apheresis is challenging in heavily pretreated patients who suffer from rapid progressive disease and receive T cell impairing medication. Methods: A total of 75 unstimulated leukaphereses were analyzed including 45 aphereses in patients and 30 in healthy donors. Thereof, 41 adult patients with Non-Hodgkin’s lymphoma (85%) or acute lymphoblastic leukemia (15%) underwent leukapheresis for CAR-T cell production. Results: Sufficient lymphocytes were harvested from all patients even from those with low peripheral lymphocyte counts of 0.18/nL. Only four patients required a second leukapheresis session. Leukapheresis products contained a median of 98 × 10⁸ (9 - 341 × 10⁸) total nucleated cells (TNC) with 38 × 10⁸ (4 - 232 × 10⁸) CD3+ T cells. Leukapheresis products from healthy donors as well as from patients in complete remission were characterized by high TNC and CD3+ T lymphocyte counts. CAR-T cell products could be manufactured for all but one patient. Conclusions: Sufficient yield of lymphocytes for CAR-T cell production is feasible also for patients with low peripheral blood counts. Up to 12–15 L blood volume should be processed in patients with absolute lymphocyte counts ≤ 1.0/nL.

Chimeric-antigen-receptor-T (CAR-T) cells are currently revolutionizing the field of cancer immunotherapy. Therefore, there is an urgent need for CAR-T cell monitoring by clinicians to assess cell expansion and persistence in patients. CAR-T cell manufacturers and researchers need to evaluate transduction efficiency and vector copy number for quality control. Here, CAR expression was analyzed in peripheral blood samples from patients and healthy donors by flow cytometry with four commercially available detection reagents and on the gene level by quantitative polymerase chain reaction (qPCR). Flow cytometric analysis of CAR expression showed higher mean CAR expression values for CD19 CAR detection reagent and the F(ab’)2 antibody than Protein L and CD19 Protein. In addition, the CD19 CAR detection reagent showed a significantly lower median background staining of 0.02% (range 0.007–0.06%) when compared to the F(ab’)2 antibody, CD19 protein and Protein L with 0.80% (range 0.47–1.58%), 0.65% (range 0.25–1.35%) and 0.73% (range 0.44–1.23%). Furthermore, flow cytometry-based CAR-T cell frequencies by CD19 CAR detection reagent showed a good correlation with qPCR results. In conclusion, quality control of CAR-T cell products can be performed by FACS and qPCR. For the monitoring of CAR-T cell frequencies by FACS in patients, CAR detection reagents with a low background staining are preferable.

Chimeric antigen receptor T (CAR-T) cell therapy has proven to be very effective in patients with relapsed/refractory acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma (NHL). However, infections—related either due to lymphodepletion or the CAR-T cell therapy itself—can result in severe and potentially life-threatening complications, while side effects such as cytokine release syndrome (CRS) might complicate differential diagnosis. Sixty-seven dosings of CAR-T cells in sixty adult patients with NHL (85%) and ALL (15%) receiving CAR-T cell therapy were assessed for infectious complications. Almost two-thirds of patients (61%) developed fever following lymphodepletion and CAR-T cell dosing. Microbiological or radiological findings were observed in 25% of all cases (bacterial 12%, viral 5%, fungal 8%). Inpatient infections were associated with more lines of therapy and more severe CRS. However, overall serious complications were rare after CAR-T therapy, with one patient dying of infection. Pathogen detection after inpatient stay was infrequent and mostly occurred in the first 90 days after dosing. Infections in CAR-T cell treated patents are common. Fast and suitable identification and treatment are crucial in these heavily pretreated and immunocompromised patients. In most cases infectious complications are manageable. Nonetheless, standardized anti-infective prophylaxis and supportive therapy are mandatory to reduce morbidity and mortality in CAR-T cell therapy.