Search

The COVID-19 pandemic threatens patients with a compromised immune and endothelial system, including patients who underwent allogeneic stem cell transplantation (alloSCT). Thus, there is an unmet need for optimizing vaccination management in this high-risk cohort. Here, we monitored antibodies against SARS-CoV-2 spike protein (anti-S1) in 167 vaccinated alloSCT patients. Humoral immune responses were detectable in 81% of patients after two vaccinations with either mRNA-, vector-based, or heterologous regimens. Age, B-cell counts, time interval from vaccination, and the type of vaccine determined antibody titres in patients without systemic immunosuppression (sIS). Similar to a healthy control cohort, mRNA vaccine-based regimens induced higher titres than vector-based vaccines. Patients on two or more immunosuppressants rarely developed immunity. In contrast, 62% and 45% of patients without or on only one immunosuppressant, respectively, showed a strong humoral vaccination response (titre > 100). Exacerbation of cGVHD upon vaccination was observed in 6% of all patients and in 22% of patients receiving immunosuppression for cGVHD. cGVHD exacerbation and low antibody titres were both associated with higher angiopoietin-2 (ANG2) serum levels. In conclusion, mRNA-based vaccines elicit strong humoral responses in alloSCT patients in the absence of double sIS. Biomarkers such as ANG2 might help with weighing cGVHD risk versus beneficial responses. [ABSTRACT FROM AUTHOR]

Adoptive cell therapy with NY-ESO-1-specific T cells is a promising option for the treatment of soft tissue sarcoma (STS) but achieves only transient tumor control in the majority of cases. A strategy to optimize this cell therapeutic approach might be the modulation of the expression of the cancer-testis antigen NY-ESO-1 using histone deacetylase inhibitors (HDACis). In this study, the ex vivo effect of combining NY-ESO-1-specific T cells with the clinically approved pan HDACis panobinostat or vorionstat was investigated. Our data demonstrated that STS cells were sensitive to HDACis. Administration of HDACi prior to NY-ESO-1-specific T cells exerted enhanced lysis against the NY-ESO-1+ STS cell line SW982. This correlated with an increase in the NY-ESO-1 and HLA-ABC expression of SW982 cells, as well as increased CD25 expression on NY-ESO-1-specific T cells. Furthermore, the immune reactivity of NY-ESO-1-specific CD8+ T cells in terms of cytokine release was enhanced by HDACis. In summary, pretreatment with HDACis represents a potential means of enhancing the cytotoxic efficacy of NY-ESO-1-specific T cells against NY-ESO-1-positive STS. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Simple Summary: Chimeric antigen receptor T (CAR-T) cells have become clinical practice for the treatment of acute lymphoblastic leukemia and non-Hodgkin lymphoma. The aim of this retrospective study was to assess infection complications after lymphodepletion and CAR-T cell therapy. Infections were commonly detected, but manageable in most cases. Fast and appropriate identification as well as treatment were critical, especially in this very vulnerable patient group. Effective strategies to prevent infections as well as adequate medical management also include standardized prophylaxis and additional supportive therapy. 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. [ABSTRACT FROM AUTHOR]

NY-ESO-1-specific T cells have shown promising activity in the treatment of soft tissue sarcoma (STS). However, standardized protocols for their generation are limited. Particularly, cost-effectiveness considerations of cell production protocols are of importance for conducting clinical studies. In this study, two different NY-ESO-1-specific T cell production protocols were compared. Major differences between protocols 1 and 2 include culture medium, interleukin-2 and retronectin concentrations, T cell activation strategy, and the transduction process. NY-ESO-1-specific T cells generated according to the two protocols were investigated for differences in cell viability, transduction efficiency, T cell expansion, immunophenotype as well as functionality. NY-ESO-1-specific T cells showed similar viability and transduction efficiency between both protocols. Protocol 1 generated higher absolute numbers of NY-ESO-1-specific T cells. However, there was no difference in absolute numbers of NY-ESO-1-specific T cell subsets with less-differentiated phenotypes accounting for efficient in vivo expansion and engraftment. Furthermore, cells generated according to protocol 1 displayed higher capacity of TNF-α generation, but lower cytotoxic capacities. Overall, both protocols provided functional NY-ESO-1-specific T cells. However, compared to protocol 1, protocol 2 is advantageous in terms of cost-effectiveness. Cell production protocols should be designed diligently to achieve a cost-effective cellular product for further clinical evaluation. [ABSTRACT FROM AUTHOR]

Simple Summary: To monitor patients after CAR T cell treatment, measuring frequencies of chimeric antigen receptor (CAR) T cells is crucial. However, experimental assays to quantify CAR T cells are lacking. Here, we describe a quantitative single copy gene-based PCR approach to measure frequencies of CAR T cells based on the FMC63 single chain variable fragment (scFv) including commercially available CAR T cell products. Besides enabling to monitor development of CAR T cells after treatment and guide further therapeutic decisions, this quantification assay proved highly useful for diagnosis of CAR T cell associated neurotoxic side effects. Overall, this quantification approach contributes significantly to the better monitoring and safety of treatment of patients with CAR T cells. Chimeric antigen receptor (CAR) T cell (CART) therapy has been established as a treatment option for patients with CD19-positive lymphoid malignancies in both the refractory and the relapsed setting. Displaying significant responses in clinical trials, two second-generation CART products directed against CD19, axicabtagene ciloleucel (axi-cel) and tisagenlecleucel (tisa-cel), have been approved and integrated into the clinical routine. However, experimental assay for quantitative monitoring of both of these CART products in treated patients in the open domain are lacking. To address this issue, we established and validated a quantitative single copy gene (SCG)-based duplex (DP)-PCR assay (SCG-DP-PCR) to quantify CARTs based on the FMC63 single chain variable fragment (scFv), i.e., axi-cel and tisa-cel. This quantitative PCR (qPCR) approach operates without standard curves or calibrator samples, offers a tool to assess cellular kinetics of FMC63 CARTs and allows direct comparison of CART-copies in axi-cel versus tisa-cel patient samples. For treating physicians, SCG-DP-PCR is an important tool to monitor CARTs and guide clinical decisions regarding CART effects in respective patients. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor T cell (CART) therapy is currently one of the most promising treatment approaches in cancer immunotherapy. However, the immunosuppressive nature of the tumor microenvironment, in particular increased reactive oxygen species (ROS) levels, provides considerable limitations. In this study, we aimed to exploit increased ROS levels in the tumor microenvironment with prodrugs of ROS accelerators, which are specifically activated in cancer cells. Upon activation, ROS accelerators induce further generation of ROS. This leads to an accumulation of ROS in tumor cells. We hypothesized that the latter cells will be more susceptible to CARTs. CD19-specific CARTs were generated with a CD19.CAR.CD28.CD137zeta third-generation retroviral vector. Cytotoxicity was determined by chromium-51 release assay. Influence of the ROS accelerators on viability and phenotype of CARTs was determined by flow cytometry. The combination of CARTs with the ROS accelerator PipFcB significantly increased their cytotoxicity in the Burkitt lymphoma cell lines Raji and Daudi, as well as primary chronic lymphocytic leukemia cells. Exposure of CARTs to PipFcB for 48 h did not influence T cell exhaustion, viability, or T cell subpopulations. In summary, the combination of CARTs with ROS accelerators may improve adoptive immunotherapy and help to overcome tumor microenvironment-mediated treatment resistance. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]