Evaluation of all-cause mortality and cardiovascular safety in patients receiving chimeric antigen receptor T cell therapy: a prospective cohort study.

Background: Assessment of cardiovascular risk is critical for patients with cancer. Previous retrospective studies suggest potential cardiotoxicity of CAR T cell therapies. We aimed to prospectively assess cardiotoxicity and the predictive value of cardiac biomarkers and classical risk factors (age, cardiac function, diabetes, arterial hypertension, smoking) for cardiac events and all-cause mortality (ACM). Methods: In this prospective cohort study, all patients treated with CAR T cell constructs (axi-cel, tisa-cel, brexu-cel, ide-cel, or the 3rd generation CAR HD-CAR-1) from Oct 1, 2018, to Sept 30, 2022 at the University Hospital Heidelberg were included. Surveillance included cardiac assessment with biomarkers (high-sensitive Troponin T (hs-cTnT), N-terminal brain natriuretic peptide (NT-proBNP)), 12-lead-ECG, and 2D echocardiography. ACM was defined as the primary study endpoint, while cardiotoxicity, defined by clinical syndromes of heart failure or decline in ejection fraction, served as a secondary endpoint. Findings: Overall, 137 patients (median age 60, range 20-83, IQR 16), were included in the study. 46 patients died during the follow up period (median 0.75 years, range 0.02-4.33, IQR 0.89) 57 month, with a median survival of 0.57 years (range 0.03-2.38 years, IQR 0.79). A septal wall thickness above 11 mm (HR 2.48, 95%-CI = 1.10-5.67, p = 0.029) was associated with an increased risk of ACM, with a trend seen for reduced left ventricular ejection fraction prior to therapy (LVEF <40%; HR 9.17, 95%-CI = 1.30-183.11, p = 0.051). Secondary endpoint was reached by 93 patients while no baseline parameter was able to predict an elevated risk. However, hs-cTnT change from baseline of 50% or more during the first 14 days after CAR infusion predicted ACM (HR 3.81, 95%-CI = 1.58-9.45; p = 0.003). None of the baseline characteristics was able to predict the incidence of cardiac events. Interpretation: Reduced pre-lymphodepletion ejection fraction and early post-infusion biomarker kinetics may be associated with increased ACM and cardiotoxicity events. These findings may help to identify patients who could benefit from intensified cardio-oncological surveillance. Funding: The German Center for Cardiovascular Research, German Research Foundation, and the Federal Ministry of Education and Research.

Extracorporeal photopheresis as a promising strategy for the treatment of GvHD after CAR-T cell therapy.

Graft-versus-host disease (GvHD) occurs in about 10-33% of patients receiving "al-logeneic" or "autologous" CAR-T cells after preceding allogeneic hematopoietic stem cell transplantation (allo-HSCT) due to the substantial presence of alloreactive T cells. Extracorporeal photopheresis (ECP) shows promising clinical outcomes in the treatment of GvHD after allo-HSCT without hampering anti-tumor and anti-viral effects. This raises an interesting question: whether ECP might constitute a new way to treat patients with GvHD after CAR-T cell therapy without compromising CAR-T cells significantly. Third-generation CD19-specific CAR-T cells were generated and an in vitro ECP protocol was established. The impact of ECP on CAR-T cells was comprehensively investigated in two models: the non-dilution model reflects days following CAR-T cell infusion and the dilution model weeks after infusion. The ther-apeutic effect of ECP on GvHD was examined in an in vitro mixed lymphocyte reac-tion (MLR) assay. We found out that ECP treated CAR-T cells demonstrated reduced potency in inducing alloreaction compared to the group without ECP treatment in MLR assay. ECP could selectively induce apoptosis, thereby enriching the naive and central memory CAR-T cells with a reduced alloreactivity. The cytokine milieu of CAR-T cells could be switched from immune stimulation to immune tolerance in both models. Moreover, ECP could modulate the proliferative capacity of CAR-T cells without hampering their long-term functionality in the dilution model. In con-clusion, ECP constitutes a promising treatment strategy for GvHD after allo-HSCT and CAR-T cell transfusion, as ECP reduces the alloreactivity without hampering CAR-T cell functionality.

In Vitro Functionality and Endurance of GMP-Compliant Point-of-Care BCMA.CAR-T Cells at Different Timepoints of Cryopreservation.

The search for target antigens for CAR-T cell therapy against multiple myeloma defined the B-cell maturation antigen (BCMA) as an interesting candidate. Several studies with BCMA-directed CAR-T cell therapy showed promising results. Second-generation point-of-care BCMA.CAR-T cells were manufactured to be of a GMP (good manufacturing practice) standard using the CliniMACS Prodigy® device. Cytokine release in BCMA.CAR-T cells after stimulation with BCMA positive versus negative myeloma cell lines, U266/HL60, was assessed via intracellular staining and flow cytometry. The short-term cytotoxic potency of CAR-T cells was evaluated by chromium-51 release, while the long-term potency used co-culture (3 days/round) at effector/target cell ratios of 1:1 and 1:4. To evaluate the activation and exhaustion of CAR-T cells, exhaustion markers were assessed via flow cytometry. Stability was tested through a comparison of these evaluations at different timepoints: d0 as well as d + 14, d + 90 and d + 365 of cryopreservation. As results, (1) Killing efficiency of U266 cells correlated with the dose of CAR-T cells in a classical 4 h chromium-release assay. There was no significant difference after cryopreservation on different timepoints. (2) In terms of endurance of BCMA.CAR-T cell function, BCMA.CAR-T cells kept their ability to kill all tumor cells over six rounds of co-culture. (3) BCMA.CAR-T cells released high amounts of cytokines upon stimulation with tumor cells. There was no significant difference in cytokine release after cryopreservation. According to the results, BCMA.CAR-T cells manufactured under GMP conditions exerted robust and specific killing of target tumor cells with a high release of cytokines. Even after 1 year of cryopreservation, cytotoxic functions were maintained at the same level. This gives clinicians sufficient time to adjust the timepoint of BCMA.CAR-T cell application to the patient's course of the underlying disease.

PD-1 checkpoint inhibition enhances the antilymphoma activity of CD19-CAR-iNKT cells that retain their ability to prevent alloreactivity.

BACKGROUND: Relapse and graft-versus-host disease (GVHD) are the main causes of death after allogeneic hematopoietic cell transplantation (HCT). Preclinical murine models and clinical data suggest that invariant natural killer T (iNKT) cells prevent acute and chronic GVHD. In addition, iNKT cells are crucial for efficient immune responses against malignancies and contribute to reduced relapse rates after transplantation. Chimeric antigen receptors (CAR) redirect effector cells to cell surface antigens and enhance killing of target cells. With this study, we aimed to combine enhanced cytotoxicity of CD19-CAR-iNKT cells against lymphoma cells with their tolerogenic properties. METHODS: iNKT cells were isolated from peripheral blood mononuclear cells and transduced with an anti-CD19-CAR retrovirus. After in vitro expansion, the functionality of CD19-CAR-iNKT cells was assessed by flow cytometry, image stream analysis and multiplex analysis in single-stimulation or repeated-stimulation assays. Moreover, the immunoregulatory properties of CD19-CAR-iNKT cells were analyzed in apoptosis assays and in mixed lymphocyte reactions. The effect of checkpoint inhibition through nivolumab was analyzed in these settings. RESULTS: In this study, we could show that the cytotoxicity of CD19-CAR-iNKT cells was mediated either through engagement of their CAR or their invariant T-cell receptor, which may circumvent loss of response through antigen escape. However, encounter of CD19-CAR-iNKT cells with their target induced a phenotype of exhaustion. Consequently, checkpoint inhibition increased cytokine release, cytotoxicity and survival of CD19-CAR-iNKT cells. Additionally, they showed robust suppression of alloreactive immune responses. CONCLUSION: In this work, we demonstrate that CAR-iNKT cells are a powerful cytotherapeutic option to prevent or treat relapse while potentially reducing the risk of GVHD after allogeneic HCT.

Stem cell collection after lenalidomide, bortezomib and dexamethasone plus elotuzumab or isatuximab in newly diagnosed multiple myeloma patients: a single centre experience from the GMMG-HD6 and -HD7 trials.

BACKGROUND: While quadruplet induction therapies deepen responses in newly diagnosed multiple myeloma patients, their impact on peripheral blood stem cell (PBSC) collection remains incompletely understood. This analysis aims to evaluate the effects of prolonged lenalidomide induction and isatuximab- or elotuzumab-containing quadruplet induction therapies on PBSC mobilization and collection. METHODS: A total of 179 transplant-eligible patients with newly diagnosed MM treated at a single academic center were included. The patients were evaluated based on PBSC mobilization and collection parameters, including overall collection results, CD34+ cell levels in peripheral blood, leukapheresis (LP) delays, overall number of LP sessions, and the rate of rescue mobilization with plerixafor. The patients underwent four different induction regimens: Lenalidomide, bortezomib, and dexamethasone (RVd, six 21-day cycles, n = 44), isatuximab-RVd (six 21-day cycles, n = 35), RVd (four 21-day cycles, n = 51), or elotuzumab-RVd (four 21-day cycles, n = 49). RESULTS: The patients' characteristics were well balanced across the different groups. Collection failures, defined as the inability to collect three sufficient PBSC transplants, were rare (n = 3, 2%), with no occurrences in the isatuximab-RVd and elotuzumab-RVd groups. Intensified induction with six 21-day cycles of RVd did not negatively impact the overall number of collected PBSCs (9.7 × 106/kg bw versus 10.5 × 106/kg bw, p = 0.331) compared to four 21-day cycles of RVd. Plerixafor usage was more common after six cycles of RVd compared to four cycles (16% versus 8%). Addition of elotuzumab to RVd did not adversely affect overall PBSC collection (10.9 × 106/kg bw versus 10.5 × 106/kg bw, p = 0.915). Patients treated with isatuximab-RVd (six cycles) had lower numbers of collected stem cells compared to those receiving RVd (six cycles) induction (8.8 × 106/kg bw versus 9.7 × 106/kg bw, p = 0.801), without experiencing significant delays in LP or increased numbers of LP sessions in a multivariable logistic regression analysis. Plerixafor usage was more common after isatuximab plus RVd compared to RVd alone (34% versus 16%). CONCLUSIONS: This study demonstrates that stem cell collection is feasible after prolonged induction with isatuximab-RVd without collection failures and might be further explored as induction therapy. TRIAL REGISTRATION: Patients were treated within the randomized phase III clinical trials GMMG-HD6 (NCT02495922, 24/06/2015) and GMMG-HD7 (NCT03617731, 24/07/2018). However, during stem cell mobilization and -collection, no study-specific therapeutic intervention was performed.

Autologous Stem Cell Collection after Daratumumab, Bortezomib, Thalidomide, and Dexamethasone versus Bortezomib, Cyclophosphamide, and Dexamethasone in Newly Diagnosed Multiple Myeloma.

Introduction: In transplant-eligible, newly diagnosed multiple myeloma (NDMM) patients, autologous peripheral blood stem cell (PBSC) collection is usually pursued after induction therapy. While induction regimens are constantly refined regarding response, their impact on PBSC collection is not fully studied. The inclusion of the anti-CD38 antibody daratumumab into induction therapy significantly improved outcomes for patients with NDMM, e.g., as part of the daratumumab, bortezomib, thalidomide, and dexamethasone (Dara-VTD) protocol. Preliminary data from the phase 3 CASSIOPEIA study proved the efficacy of Dara-VTD. While overall PBSC collection upon addition of daratumumab was reduced in the study population, more detailed analyses on the impact are missing. Methods: We here report on PBSC mobilization and collection metrics in n = 119 patients with NDMM who underwent induction therapy with bortezomib, cyclophosphamide, and dexamethasone (VCD, n = 61) or Dara-VTD (n = 58). Results: Patient characteristics were well balanced between groups. The Dara-VTD group showed improved response parameters with 66% of patients reaching at least very good partial response versus 54% in the VCD group. Dara-VTD patients exhibited inferior mobilization metrics such as peripheral blood CD34+ cell count at the first leukapheresis (LP) session (65 vs. 106/µL, p = 0.001), median number of LP sessions (2 vs. 1, p = 0.001), and PBSC collection at first LP (5.5 vs. 8.3 × 106/kg body weight [bw], p = 0.001). Utilization of plerixafor was slightly higher after Dara-VTD (33% vs. 21% of patients, p = 0.143). The overall PBSC collection result was significantly lower after Dara-VTD (8.4 vs. 9.6 × 106/kg bw, p = 0.026). 78% and 85% of patients successfully collected 3 transplants with ≥2 × 106 CD34+ cells/kg bw in the Dara-VTD and the VCD groups, respectively. Conclusion: In summary, Dara-VTD, possibly due to both anti-CD38 antibody and thalidomide exposure, imposes a limitation on PBSC collection which can be only partly overcome by utilization of plerixafor.

First third-generation CAR T cell application targeting CD19 for the treatment of systemic IgM AL amyloidosis with underlying marginal zone lymphoma.

Light chain amyloidosis (AL) is a rare disease caused by the generalized deposition of misfolded free light chains. Patients with immunoglobulin M gammopathy (IgM) and indolent B-cell lymphoma such as marginal zone lymphoma (MZL) may in some instances develop AL amyloidosis. So far, CAR T cells for AL amyloidosis have only been reported utilizing the B cell maturation antigen as target, while CD19 has so far not been used in AL amyloidosis.We report the case of a 71-year-old male, diagnosed with systemic AL kappa amyloidosis and MZL, receiving third-generation CAR T cell therapy targeting CD19. Prior treatment included bendamustine/rituximab and cyclophosphamide/ dexamethasone with subsequent autologous stem cell transplantation. CAR T application was well tolerated despite heart and kidney amyloid manifestations, and only early low-grade procedure-specific toxicities were observed. A continuous decrease in IgM, kappa light chains and kappa-to-lambda light chain difference was observed in the patient from day + 30 on, resulting in a deep hematological response six months after treatment.In summary, we present a novel case of CAR T cell treatment with third generation CD19 directed infusion for AL amyloidosis with an underlying secretory active B cell lymphoma, showing that this is an effective treatment modality and can be applied to patients with subsequent AL amyloidosis.

Impact of Clinical Parameters and Induction Regimens on Peripheral Blood Stem-Cell Mobilization and Collection in Multiple Myeloma Patients.

Introduction: High-dose chemotherapy (HDCT) followed by autologous blood stem-cell transplantation (ABSCT) remains the standard consolidation therapy for newly diagnosed eligible multiple myeloma (MM) patients. As a prerequisite, peripheral blood stem cells (PBSCs) must be mobilized and collected by leukapheresis (LP). Many factors can hamper PBSC mobilization/collection. Here, we provide a comprehensive multiparametric assessment of PBSC mobilization/collection outcome parameters in a large cohort. Methods: In total, 790 MM patients (471 [60%] male, 319 [40%] female) who underwent PBSC mobilization/collection during first-line treatment were included. Evaluated PBSC mobilization/collection outcome parameters included the prolongation of PBSC mobilization, plerixafor administration, number of LP sessions, and overall PBSC collection goal/result. Results: 741 (94%) patients received cyclophosphamide/adriamycin/dexamethasone (CAD) and granulocyte-colony-stimulating factor (G-CSF) mobilization. Plerixafor was administered in 80 (10%) patients. 489 (62%) patients started LP without delay. 530 (67%) patients reached the PBSC collection goal at the first LP session. The mean overall PBSC collection result was 10.3 (standard deviation [SD] 4.4) × 106 CD34+ cells/kg. In a multiparametric analysis, variables negatively associated with PBSC mobilization/collection outcomes were female gender, age >60 years, an advanced ISS stage, and local radiation pre-/during induction, but not remission status postinduction. Notably, the identified risk factors contributed differently to each PBSC mobilization/collection outcome parameter. In this context, compared to all other induction regimens, lenalidomide-based induction with/without antibodies negatively affected only the number of LP sessions required to reach the collection goal, but no other PBSC mobilization/collection outcome parameters. In contrast, the probability of reaching a high collection goal of ≥6 × 106 CD34+ cells/kg body weight was higher after lenalidomide-based induction compared to VCD/PAD or VAD - taking into account - that a higher G-SCF dosage was given in approximately one-third of patients receiving lenalidomide-based induction with/without antibodies. Conclusion: Considering the identified risk factors in the clinical setting can contribute to optimized PBSC mobilization/collection. Moreover, our study demonstrates the necessity for a differentiated evaluation of PBSC mobilization/collection outcome parameters.

Five-year follow-up of a phase I trial of donor-derived modified immune cell infusion in kidney transplantation.

Background: The administration of modified immune cells (MIC) before kidney transplantation led to specific immunosuppression against the allogeneic donor and a significant increase in regulatory B lymphocytes. We wondered how this approach affected the continued clinical course of these patients. Methods: Ten patients from a phase I clinical trial who had received MIC infusions prior to kidney transplantation were retrospectively compared to 15 matched standard-risk recipients. Follow-up was until year five after surgery. Results: The 10 MIC patients had an excellent clinical course with stable kidney graft function, no donor-specific human leukocyte antigen antibodies (DSA) or acute rejections, and no opportunistic infections. In comparison, a retrospectively matched control group receiving standard immunosuppressive therapy had a higher frequency of DSA (log rank P = 0.046) and more opportunistic infections (log rank P = 0.033). Importantly, MIC patients, and in particular the four patients who had received the highest cell number 7 days before surgery and received low immunosuppression during follow-up, continued to show a lack of anti-donor T lymphocyte reactivity in vitro and high CD19+CD24hiCD38hi transitional and CD19+CD24hiCD27+ memory B lymphocytes until year five after surgery. Conclusions: MIC infusions together with reduced conventional immunosuppression were associated with good graft function during five years of follow-up, no de novo DSA development and no opportunistic infections. In the future, MIC infusions might contribute to graft protection while reducing the side effects of immunosuppressive therapy. However, this approach needs further validation in direct comparison with prospective controls. Trial registration: https://clinicaltrials.gov/, identifier NCT02560220 (for the TOL-1 Study). EudraCT Number: 2014-002086-30.

Treatment of adult ALL patients with third-generation CD19-directed CAR T cells: results of a pivotal trial.

BACKGROUND: Third-generation chimeric antigen receptor (CAR)-engineered T cells (CARTs) might improve clinical outcome of patients with B cell malignancies. This is the first report on a third-generation CART dose-escalating, phase-1/2 investigator-initiated trial treating adult patients with refractory and/or relapsed (r/r) acute lymphoblastic leukemia (ALL). METHODS: Thirteen patients were treated with escalating doses of CD19-directed CARTs between 1 × 106 and 50 × 106 CARTs/m2. Leukapheresis, manufacturing and administration of CARTs were performed in-house. RESULTS: For all patients, CART manufacturing was feasible. None of the patients developed any grade of Immune effector cell-associated neurotoxicity syndrome (ICANS) or a higher-grade (≥ grade III) catokine release syndrome (CRS). CART expansion and long-term CART persistence were evident in the peripheral blood (PB) of evaluable patients. At end of study on day 90 after CARTs, ten patients were evaluable for response: Eight patients (80%) achieved a complete remission (CR), including five patients (50%) with minimal residual disease (MRD)-negative CR. Response and outcome were associated with the administered CART dose. At 1-year follow-up, median overall survival was not reached and progression-free survival (PFS) was 38%. Median PFS was reached on day 120. Lack of CD39-expression on memory-like T cells was more frequent in CART products of responders when compared to CART products of non-responders. After CART administration, higher CD8 + and γδ-T cell frequencies, a physiological pattern of immune cells and lower monocyte counts in the PB were associated with response. CONCLUSION: In conclusion, third-generation CARTs were associated with promising clinical efficacy and remarkably low procedure-specific toxicity, thereby opening new therapeutic perspectives for patients with r/r ALL. Trial registration This trial was registered at www. CLINICALTRIALS: gov as NCT03676504.

Impact of serum­free media on the expansion and functionality of CD19.CAR T­cells.

Fetal bovine serum (FBS) or human serum is widely used in the production of chimeric antigen receptor (CAR) T­cells. In order to overcome a lot­to­lot inconsistency, the use of chemically defined medium that is free of animal-components would be highly desirable. The present study compared three serum­free media [Prime­XV™ T Cell CDM, Fujifilm™ (FF), LymphoONE™ T­Cell Expansion Xeno­Free Medium, Takara Bio™ (TB) and TCM GMP­Prototype, CellGenix™ (CG)] to the standard CAR T­cell medium containing FBS (RCF). After 12 days of CD19.CAR T­cell culture, the expansion, viability, transduction efficiency and phenotype were assessed using flow cytometry. The functionality of CAR T­cells was evaluated using intracellular staining, a chromium release assay and a long­term co­culture assay. Expansion and viability did not differ between the CAR T­cells generated in serum­free media compared to the standard FBS­containing medium. The CG CAR T­cells had a statistically significant higher frequency of IFNγ+ and IFNγ+TNF­α+ CAR T­cells than the CAR T­cells cultured with FBS (22.5 vs. 7.6%, P=0.0194; 15.3 vs. 6.2%, P=0.0399, respectively) as detected by intracellular cytokine staining. The CAR T­cells generated with serum­free media exhibited a higher cytotoxicity than the CAR T­cells cultured with FBS in the evaluation by chromium release assay [CG vs. RCF (P=0.0182), FF vs. RCF (P=0.0482) and TB vs. RCF (P=0.0482)]. Phenotyping on day 12 of CAR T­cell production did not reveal a significant difference in the expression of the exhaustion markers, programmed cell death protein 1, lymphocyte­activation gene 3 and T­cell immunoglobulin and mucin­domain containing­3. The CAR T­cells cultured in FF had a higher percentage of central memory CAR T­cells (40.0 vs. 14.3%, P=0.0470) than the CAR T­cells cultured with FBS, whereas the CAR T­cells in FF (6.2 vs. 24.2%, P=0.0029) and CG (11.0% vs. 24.2%, P=0.0468) had a lower frequency of naïve CAR T­cells. On the whole, the present study demonstrates that in general, the functionality and expansion of CAR T cells are maintained in serum­free media. Given the advantages of freedom from bovine material and consistent quality, serum­free media hold promise for the future development of the field of GMP manufacturing of CAR T­cells.

Idecabtagene Vicleucel Is Well Tolerated and Effective in Relapsed/Refractory Myeloma Patients with Prior Allogeneic Stem Cell Transplantation.

BCMA-specific chimeric-antigen receptor (CAR-) T cell therapy has led to high response rates and durable remissions in patients with relapsed refractory multiple myeloma. However, little data are available for patients after prior allogeneic stem cell transplantation (allo-SCT) in whom T cells are chimeric. In this study, we aimed to assess the safety and efficacy of patient-derived donor CAR-T therapy in myeloma patients with prior allo-SCT, particularly with regard to graft-versus-host disease (GVHD). We report a comprehensive clinical analysis of 3 patients who had previously undergone allo-SCT for high-risk myeloma and were treated with idecabtagene vicleucel (ide-cel) at our institution. Ide-cel was well tolerated, with no clinically relevant immune effector cell-associated neurotoxicity syndrome or cytokine release syndrome observed in any patient. Importantly, no new GVHD was observed, even though all patients had a history of GVHD. All patients responded to treatment with at least a very good partial remission. Two patients relapsed within 6 months, and 1 patient was still in stringent complete remission at the time of this report. Our findings demonstrate that treatment with ide-cel is feasible, very well tolerated, and effective in patients with relapsed/refractory multiple myeloma after prior allo-SCT.

A non-antibiotic-disrupted gut microbiome is associated with clinical responses to CD19-CAR-T cell cancer immunotherapy.

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.

Definition and Characterization of SOX11-Derived T Cell Epitopes towards Immunotherapy of Glioma.

The transcription factor SOX11 is a tumor-associated antigen with low expression in normal cells, but overexpression in glioblastoma (GBM). So far, conventional surgery, chemotherapy, and radiotherapy have not substantially improved the dismal prognosis of relapsed/refractory GBM patients. Immunotherapy is considered a promising strategy against GBM, but there is a fervent need for better immunotargets in GBM. To this end, we performed an in silico prediction study on SOX11, which primarily yielded ten promising HLA-A*0201-restricted peptides derived from SOX11. We defined a novel peptide FMACSPVAL, which had the highest score according to in silico prediction (6.02 nM by NetMHC-4.0) and showed an exquisite binding affinity to the HLA-A*0201 molecule in the peptide-binding assays. In the IFN-γ ELISPOT assays, FMACSPVAL demonstrated a high efficiency for generating SOX11-specific CD8+ T cells. Nine out of thirty-two healthy donors showed a positive response to SOX11, as assessed by the ELISPOT assays. Therefore, this novel antigen peptide epitope seems to be promising as a target for T cell-based immunotherapy in GBM. The adoptive transfer of in vitro elicited SOX11-specific CD8+ T cells constitutes a potential approach for the treatment of GBM patients.

Anti-SARS-CoV-2 antibody-containing plasma improves outcome in patients with hematologic or solid cancer and severe COVID-19: a randomized clinical trial.

Patients with cancer are at high risk of severe coronavirus disease 2019 (COVID-19), with high morbidity and mortality. Furthermore, impaired humoral response renders severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines less effective and treatment options are scarce. Randomized trials using convalescent plasma are missing for high-risk patients. Here, we performed a randomized, open-label, multicenter trial ( https://www.clinicaltrialsregister.eu/ctr-search/trial/2020-001632-10/DE ) in hospitalized patients with severe COVID-19 (n = 134) within four risk groups ((1) cancer (n = 56); (2) immunosuppression (n = 16); (3) laboratory-based risk factors (n = 36); and (4) advanced age (n = 26)) randomized to standard of care (control arm) or standard of care plus convalescent/vaccinated anti-SARS-CoV-2 plasma (plasma arm). No serious adverse events were observed related to the plasma treatment. Clinical improvement as the primary outcome was assessed using a seven-point ordinal scale. Secondary outcomes were time to discharge and overall survival. For the four groups combined, those receiving plasma did not improve clinically compared with those in the control arm (hazard ratio (HR) = 1.29; P = 0.205). However, patients with cancer experienced a shortened median time to improvement (HR = 2.50; P = 0.003) and superior survival with plasma treatment versus the control arm (HR = 0.28; P = 0.042). Neutralizing antibody activity increased in the plasma cohort but not in the control cohort of patients with cancer (P = 0.001). Taken together, convalescent/vaccinated plasma may improve COVID-19 outcomes in patients with cancer who are unable to intrinsically generate an adequate immune response.

Induction of Long-Lasting Regulatory B Lymphocytes by Modified Immune Cells in Kidney Transplant Recipients.

BACKGROUND: We recently demonstrated that donor-derived modified immune cells (MICs)-PBMCs that acquire immunosuppressive properties after a brief treatment-induced specific immunosuppression against the allogeneic donor when administered before kidney transplantation. We found up to a 68-fold increase in CD19 + CD24 hi CD38 hi transitional B lymphocytes compared with transplanted controls. METHODS: Ten patients from a phase 1 clinical trial who had received MIC infusions before kidney transplantation were followed to post-transplant day 1080. RESULTS: Patients treated with MICs had a favorable clinical course, showing no donor-specific human leukocyte antigen antibodies or acute rejections. The four patients who had received the highest dose of MICs 7 days before surgery and were on reduced immunosuppressive therapy showed an absence of in vitro lymphocyte reactivity against stimulatory donor blood cells, whereas reactivity against third party cells was preserved. In these patients, numbers of transitional B lymphocytes were 75-fold and seven-fold higher than in 12 long-term survivors on minimal immunosuppression and four operationally tolerant patients, respectively ( P <0.001 for both). In addition, we found significantly higher numbers of other regulatory B lymphocyte subsets and a gene expression signature suggestive of operational tolerance in three of four patients. In MIC-treated patients, in vitro lymphocyte reactivity against donor blood cells was restored after B lymphocyte depletion, suggesting a direct pathophysiologic role of regulatory B lymphocytes in donor-specific unresponsiveness. CONCLUSIONS: These results indicate that donor-specific immunosuppression after MIC infusion is long-lasting and associated with a striking increase in regulatory B lymphocytes. Donor-derived MICs appear to be an immunoregulatory cell population that when administered to recipients before transplantation, may exert a beneficial effect on kidney transplants. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: MIC Cell Therapy for Individualized Immunosuppression in Living Donor Kidney Transplant Recipients (TOL-1), NCT02560220.

Symptomatic Patients with Hyperleukocytic FLT3-ITD Mutated Acute Myeloid Leukemia Might Benefit from Leukapheresis.

PURPOSE: We aimed to identify subsets of patients who benefit from emergency LA and to establish a therapeutic algorithm for AML patients with hyperleukocytosis. METHODS: In this single-center retrospective cohort study, a total of 20 consecutive patients underwent LA because of their clinical symptoms. Overall survival (OS) analysis was conducted using the Kaplan-Meier plot method. Univariate and multivariate analyses (using multiple logistic regression) were performed. At the time of diagnosis, all patients received a standard diagnostic workup for AML including FLT3-ITD mutational analysis. RESULTS: FLT3-ITD mut AML patients receiving LA had a median OS of 437 days (range 5-2379 days) with a corresponding 14-day survival of 92.3%, while FLT3 wt AML patients displayed a significantly lower median OS of only 5 days (range 1-203 days) with a corresponding 14-day survival of 14.3% (p = 0.0001). CONCLUSIONS: Among patients with clinical symptoms of leukostasis, the subset of FLT3-ITD mut AML patients showed a better outcome with lower early mortality after emergency LA. Based on these observations, we established a therapeutic algorithm for AML patients with hyperleukocytosis.

Individualised immunosuppression with intravenously administered donor-derived modified immune cells compared with standard of care in living donor kidney transplantation (TOL-2 Study): protocol for a multicentre, open-label, phase II, randomised controlled trial.

INTRODUCTION: Donor-derived modified immune cells (MIC) induced long-term specific immunosuppression against the allogeneic donor in preclinical models of transplantation. In a phase I clinical trial (TOL-1 Study), MIC treatment resulted in a cellular phenotype that was directly and indirectly suppressive to the recipient's immune system allowing for reduction of conventional immunosuppressive therapy. Here, we describe a protocol for a randomised controlled, multicentre phase-IIb clinical trial of individualised immunosuppression with intravenously administered donor MIC compared with standard-of-care (SoC) in living donor kidney transplantation (TOL-2 Study). METHODS AND ANALYSIS: Sixty-three living donor kidney transplant recipients from six German transplant centres are randomised 2:1 to treatment with MIC (MIC group, N=42) or no treatment with MIC (control arm, N=21). MIC are manufactured from donor peripheral blood mononuclear cells under Good Manufacturing Practice conditions. The primary objective of this trial is to determine the efficacy of MIC treatment together with reduced conventional immunosuppressive therapy in terms of achieving an operational tolerance-like phenotype compared with SoC 12 months after MIC administration. Key secondary endpoints are the number of patient-relevant infections as well as a composite of biopsy-proven acute rejection, graft loss, graft dysfunction or death. Immunosuppressive therapy of MIC-treated patients is reduced during follow-up under an extended immunological monitoring including human leucocyte antigen-antibody testing, and determination of lymphocyte subsets, for example, regulatory B lymphocytes (Breg) and antidonor T cell response. A Data Safety Monitoring Board has been established to allow an independent assessment of safety and efficacy. ETHICS AND DISSEMINATION: Ethical approval has been provided by the Ethics Committee of the Medical Faculty of the University of Heidelberg, Heidelberg, Germany (AFmu-580/2021, 17 March 2022) and from the Federal Institute for Vaccines and Biomedicines, Paul-Ehrlich-Institute, Langen, Germany (Vorlage-Nr. 4586/02, 21 March 2022). Written informed consent will be obtained from all patients and respective donors prior to enrolment in the study. The results from the TOL-2 Study will be published in peer-reviewed medical journals and will be presented at symposia and scientific meetings. TRIAL REGISTRATION NUMBER: NCT05365672.

Comparison of single copy gene­based duplex quantitative PCR and digital droplet PCR for monitoring of expansion of CD19­directed CAR T cells in treated patients.

Chimeric antigen receptor (CAR) T cell therapy with axicabtagene ciloleucel, tisagenlecleucel and brexucabtagen ciloleucel has been adopted as the standard of care for patients with refractory and/or relapsed CD19­positive lymphoid malignancies. Monitoring of kinetics of CAR T cells after administration is crucial for patient follow­up and important to guide clinical decisions for patients subjected to CAR T cell therapy. Information of transgene copies within a CAR T cell product prior to administration, i.e. vector copy numbers, is of high importance to warrant patient safety. However, experimental assays for quantitative CAR T cell monitoring in the open domain are currently lacking. Several institutions have established in­house assays to monitor CAR T cell frequencies. In the present study, the quantitative (q)PCR assay established at the Heidelberg University Hospital (Heidelberg, Germany), i.e. single copy gene­based duplex qPCR, was compared with the digital droplet PCR assay established at the University Medical Center Hamburg­Eppendorf (Hamburg, Germany). Both methods that were independently developed enable accurate and comparable CAR T cell frequency assessment and are useful in the clinical setting.